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Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides.

Gao J, Wymore RS, Wang Y, Gaudette GR, Krukenkamp IB, Cohen IS, Mathias RT - J. Gen. Physiol. (2002)

Bottom Line: 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 the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the alpha(2)-isoform, but both the stimulatory and inhibitory concentrations of ouabain were approximately 10-fold lower than those for DHO.These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I(P) in heart by nanomolar concentrations of endogenous ouabain-like molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics and Institute of Molecular Cardiology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA.

ABSTRACT
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(+)](o), as was I(P); (2) it was absent in 0 mM [Na(+)](i), as was I(P); (3) at reduced [Na(+)](i), the outward current was reduced in proportion to the reduction in I(P); (4) it was eliminated by intracellular vanadate, as was I(P). Our previous work suggested guinea pig ventricular myocytes coexpress the alpha(1)- and alpha(2)-isoforms of the Na/K pumps. The stimulation of I(P) appears to be through stimulation of the high glycoside affinity alpha(2)-isoform and not the alpha(1)-isoform because of the following: (1) regulatory signals that specifically increased activity of the alpha(2)-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the alpha(1)-isoform did not affect the stimulation; (3) changes in [K(+)](o) that affected activity of the alpha(1)-isoform, but not the alpha(2)-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the alpha(1)-isoform but not the alpha(2)-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total I(P) increased by 35 +/- 10% (mean +/- SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the alpha(2)-isoform, then activity of the alpha(2)-isoform increased by 107 +/- 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the alpha(2)-isoform, but both the stimulatory and inhibitory concentrations of ouabain were approximately 10-fold lower than those for DHO. Stimulation of I(P) by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the alpha(1)- and alpha(3)-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the alpha(3)-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of I(P) that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of I(P), and where the contributions of the high glycoside affinity alpha(2)- and alpha(3)-isoforms could be separated from that of the alpha(1)-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I(P) in heart by nanomolar concentrations of endogenous ouabain-like molecules.

Show MeSH
Stimulation of IP in guinea pig ventricular myocytes. (A) Slow superfusion of solution containing 5 μM DHO caused the bath concentration of DHO to slowly rise from 0 to 5 μM. This induced an initial outward transient in holding current (arrow) followed by the inward shift. The outward transient was not observed when 5 μM DHO was rapidly superfused. See results for details. (B) The steady state increase in outward holding current induced by 10 nM DHO in the presence of 5.4 mM [K+]o did not occur when the external K+ was removed, suggesting the outward shift in current was due to stimulation of IP. (C) Stimulation of IP was intracellular Na+-dependent. (top) Intracellular Na+ was completely removed after waiting 6 min in the whole-cell mode with a pipette resistance (RP) of 1.5 MΩ. Neither the outward shift in current at 10 nM DHO nor inhibition of IP by 1 mM DHO was observed. (middle) In a second experiment, a small amount of Na+ remained in the cell after waiting only 5 min with RP of 4 MΩ. A small shift in outward current at 10 nM DHO and a small inhibition of IP by 1 mM DHO were observed. (bottom) In a third experiment, a larger amount of Na+ remained in the cell after waiting 4 min with RP of 5 MΩ. A larger outward shift in current and a larger blockade of IP were observed. Thus, the outward shift in current at 10 nM DHO and blockade of IP at 1 mM DHO were highly correlated, providing further evidence that the outward current was due to stimulation of IP. (D) Intracellular vanadate blocks both IP and the low DHO-dependent outward current. (left) When 1 mM sodium orthovanadate was included in the pipette solution, 10 nM DHO did not cause an outward shift in current and 1 mM DHO did not cause an inward shift in current, indicating inhibition of total IP also inhibited the low DHO stimulation of IP. (right) Cells isolated from ventricles of the same heart show a typical stimulation and inhibition of IP by low and high [DHO], respectively, when vanadate was not in the pipette solution.
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fig2: Stimulation of IP in guinea pig ventricular myocytes. (A) Slow superfusion of solution containing 5 μM DHO caused the bath concentration of DHO to slowly rise from 0 to 5 μM. This induced an initial outward transient in holding current (arrow) followed by the inward shift. The outward transient was not observed when 5 μM DHO was rapidly superfused. See results for details. (B) The steady state increase in outward holding current induced by 10 nM DHO in the presence of 5.4 mM [K+]o did not occur when the external K+ was removed, suggesting the outward shift in current was due to stimulation of IP. (C) Stimulation of IP was intracellular Na+-dependent. (top) Intracellular Na+ was completely removed after waiting 6 min in the whole-cell mode with a pipette resistance (RP) of 1.5 MΩ. Neither the outward shift in current at 10 nM DHO nor inhibition of IP by 1 mM DHO was observed. (middle) In a second experiment, a small amount of Na+ remained in the cell after waiting only 5 min with RP of 4 MΩ. A small shift in outward current at 10 nM DHO and a small inhibition of IP by 1 mM DHO were observed. (bottom) In a third experiment, a larger amount of Na+ remained in the cell after waiting 4 min with RP of 5 MΩ. A larger outward shift in current and a larger blockade of IP were observed. Thus, the outward shift in current at 10 nM DHO and blockade of IP at 1 mM DHO were highly correlated, providing further evidence that the outward current was due to stimulation of IP. (D) Intracellular vanadate blocks both IP and the low DHO-dependent outward current. (left) When 1 mM sodium orthovanadate was included in the pipette solution, 10 nM DHO did not cause an outward shift in current and 1 mM DHO did not cause an inward shift in current, indicating inhibition of total IP also inhibited the low DHO stimulation of IP. (right) Cells isolated from ventricles of the same heart show a typical stimulation and inhibition of IP by low and high [DHO], respectively, when vanadate was not in the pipette solution.

Mentions: After whole-cell recording was initiated, a period of at least 5 min was required for the pipette and intracellular solutions to come to steady state (Gao et al., 1992). When steady state was achieved, different concentrations of DHO were superfused to observe the DHO-induced changes in holding current. In Fig. 1 A, when 5 μM DHO was applied rapidly, an inward shift in the holding current was observed, indicating inhibition of the current generated by the α2-isoform of the Na/K pumps (Gao et al., 1999a; for review see Mathias et al., 2000). Upon washout of DHO, the holding current returned to its original level. Our first indication that nanomolar DHO might stimulate IP was observed in the same cell, when 5 μM DHO was applied slowly. In this situation, the holding current experienced an initial outward transient (indicated by the arrow), which could not be attributed to any artifact, suggesting very low concentrations of DHO may stimulate the pumps. The same results were consistently observed in a total of eight cells, suggesting relatively low concentrations of DHO might have evoked the initial increase in holding current (i.e., as the solution containing 5 μM DHO was slowly perfused into the DHO-free bath, the initial [DHO] was much lower than 5 μM). Therefore, we used 10 nM DHO to investigate whether a steady state increase in outward current could be generated (Fig. 1 B). Upon washout of DHO, the holding current returned to its original level. However, when the external K+ was removed, 10 nM DHO did not induce any change in the holding current in the same cell. The same results were observed in a total of six cells, suggesting that the steady state increase in outward current by 10 nM DHO at 5.4 mM [K+]o could be due to stimulation of IP.


Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides.

Gao J, Wymore RS, Wang Y, Gaudette GR, Krukenkamp IB, Cohen IS, Mathias RT - J. Gen. Physiol. (2002)

Stimulation of IP in guinea pig ventricular myocytes. (A) Slow superfusion of solution containing 5 μM DHO caused the bath concentration of DHO to slowly rise from 0 to 5 μM. This induced an initial outward transient in holding current (arrow) followed by the inward shift. The outward transient was not observed when 5 μM DHO was rapidly superfused. See results for details. (B) The steady state increase in outward holding current induced by 10 nM DHO in the presence of 5.4 mM [K+]o did not occur when the external K+ was removed, suggesting the outward shift in current was due to stimulation of IP. (C) Stimulation of IP was intracellular Na+-dependent. (top) Intracellular Na+ was completely removed after waiting 6 min in the whole-cell mode with a pipette resistance (RP) of 1.5 MΩ. Neither the outward shift in current at 10 nM DHO nor inhibition of IP by 1 mM DHO was observed. (middle) In a second experiment, a small amount of Na+ remained in the cell after waiting only 5 min with RP of 4 MΩ. A small shift in outward current at 10 nM DHO and a small inhibition of IP by 1 mM DHO were observed. (bottom) In a third experiment, a larger amount of Na+ remained in the cell after waiting 4 min with RP of 5 MΩ. A larger outward shift in current and a larger blockade of IP were observed. Thus, the outward shift in current at 10 nM DHO and blockade of IP at 1 mM DHO were highly correlated, providing further evidence that the outward current was due to stimulation of IP. (D) Intracellular vanadate blocks both IP and the low DHO-dependent outward current. (left) When 1 mM sodium orthovanadate was included in the pipette solution, 10 nM DHO did not cause an outward shift in current and 1 mM DHO did not cause an inward shift in current, indicating inhibition of total IP also inhibited the low DHO stimulation of IP. (right) Cells isolated from ventricles of the same heart show a typical stimulation and inhibition of IP by low and high [DHO], respectively, when vanadate was not in the pipette solution.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2238186&req=5

fig2: Stimulation of IP in guinea pig ventricular myocytes. (A) Slow superfusion of solution containing 5 μM DHO caused the bath concentration of DHO to slowly rise from 0 to 5 μM. This induced an initial outward transient in holding current (arrow) followed by the inward shift. The outward transient was not observed when 5 μM DHO was rapidly superfused. See results for details. (B) The steady state increase in outward holding current induced by 10 nM DHO in the presence of 5.4 mM [K+]o did not occur when the external K+ was removed, suggesting the outward shift in current was due to stimulation of IP. (C) Stimulation of IP was intracellular Na+-dependent. (top) Intracellular Na+ was completely removed after waiting 6 min in the whole-cell mode with a pipette resistance (RP) of 1.5 MΩ. Neither the outward shift in current at 10 nM DHO nor inhibition of IP by 1 mM DHO was observed. (middle) In a second experiment, a small amount of Na+ remained in the cell after waiting only 5 min with RP of 4 MΩ. A small shift in outward current at 10 nM DHO and a small inhibition of IP by 1 mM DHO were observed. (bottom) In a third experiment, a larger amount of Na+ remained in the cell after waiting 4 min with RP of 5 MΩ. A larger outward shift in current and a larger blockade of IP were observed. Thus, the outward shift in current at 10 nM DHO and blockade of IP at 1 mM DHO were highly correlated, providing further evidence that the outward current was due to stimulation of IP. (D) Intracellular vanadate blocks both IP and the low DHO-dependent outward current. (left) When 1 mM sodium orthovanadate was included in the pipette solution, 10 nM DHO did not cause an outward shift in current and 1 mM DHO did not cause an inward shift in current, indicating inhibition of total IP also inhibited the low DHO stimulation of IP. (right) Cells isolated from ventricles of the same heart show a typical stimulation and inhibition of IP by low and high [DHO], respectively, when vanadate was not in the pipette solution.
Mentions: After whole-cell recording was initiated, a period of at least 5 min was required for the pipette and intracellular solutions to come to steady state (Gao et al., 1992). When steady state was achieved, different concentrations of DHO were superfused to observe the DHO-induced changes in holding current. In Fig. 1 A, when 5 μM DHO was applied rapidly, an inward shift in the holding current was observed, indicating inhibition of the current generated by the α2-isoform of the Na/K pumps (Gao et al., 1999a; for review see Mathias et al., 2000). Upon washout of DHO, the holding current returned to its original level. Our first indication that nanomolar DHO might stimulate IP was observed in the same cell, when 5 μM DHO was applied slowly. In this situation, the holding current experienced an initial outward transient (indicated by the arrow), which could not be attributed to any artifact, suggesting very low concentrations of DHO may stimulate the pumps. The same results were consistently observed in a total of eight cells, suggesting relatively low concentrations of DHO might have evoked the initial increase in holding current (i.e., as the solution containing 5 μM DHO was slowly perfused into the DHO-free bath, the initial [DHO] was much lower than 5 μM). Therefore, we used 10 nM DHO to investigate whether a steady state increase in outward current could be generated (Fig. 1 B). Upon washout of DHO, the holding current returned to its original level. However, when the external K+ was removed, 10 nM DHO did not induce any change in the holding current in the same cell. The same results were observed in a total of six cells, suggesting that the steady state increase in outward current by 10 nM DHO at 5.4 mM [K+]o could be due to stimulation of IP.

Bottom Line: 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 the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the alpha(2)-isoform, but both the stimulatory and inhibitory concentrations of ouabain were approximately 10-fold lower than those for DHO.These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I(P) in heart by nanomolar concentrations of endogenous ouabain-like molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics and Institute of Molecular Cardiology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8661, USA.

ABSTRACT
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(+)](o), as was I(P); (2) it was absent in 0 mM [Na(+)](i), as was I(P); (3) at reduced [Na(+)](i), the outward current was reduced in proportion to the reduction in I(P); (4) it was eliminated by intracellular vanadate, as was I(P). Our previous work suggested guinea pig ventricular myocytes coexpress the alpha(1)- and alpha(2)-isoforms of the Na/K pumps. The stimulation of I(P) appears to be through stimulation of the high glycoside affinity alpha(2)-isoform and not the alpha(1)-isoform because of the following: (1) regulatory signals that specifically increased activity of the alpha(2)-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the alpha(1)-isoform did not affect the stimulation; (3) changes in [K(+)](o) that affected activity of the alpha(1)-isoform, but not the alpha(2)-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the alpha(1)-isoform but not the alpha(2)-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total I(P) increased by 35 +/- 10% (mean +/- SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the alpha(2)-isoform, then activity of the alpha(2)-isoform increased by 107 +/- 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the alpha(2)-isoform, but both the stimulatory and inhibitory concentrations of ouabain were approximately 10-fold lower than those for DHO. Stimulation of I(P) by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the alpha(1)- and alpha(3)-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the alpha(3)-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of I(P) that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of I(P), and where the contributions of the high glycoside affinity alpha(2)- and alpha(3)-isoforms could be separated from that of the alpha(1)-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I(P) in heart by nanomolar concentrations of endogenous ouabain-like molecules.

Show MeSH