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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.

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[K+]o modulation of IP in guinea pig ventricular myocytes suggests the α2-isoform and not the α1-isoform is involved in stimulation of IP by low [DHO]. (A) An original record of holding current showing the protocol for observing stimulation of IP by low [DHO] and inhibition of IP by high [DHO] in 4 and 8 mM [K+]o. In this cell, when [K+]o was 4 mM, the stimulation of IP was 26 pA and the inhibition was 71 pA. When [K+]o was increased to 8 mM, the stimulation of IP was 27 pA and the inhibition was 96 pA. Thus activity of the α1-isoform increased, but the stimulation did not. (B) Average results from five cells. In each cell, the stimulation by 10 nM DHO and the inhibition by 1 mM DHO were recorded in 4 and 8 mM [K+]o, and then the ratio of the stimulation of IP (1.04 ± 0.08, P = 0.31) at the two [K+]o and the ratio of the total IP (1.39 ± 0.20, P = 0.029) at the two [K+]o were recorded and averaged.
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fig5: [K+]o modulation of IP in guinea pig ventricular myocytes suggests the α2-isoform and not the α1-isoform is involved in stimulation of IP by low [DHO]. (A) An original record of holding current showing the protocol for observing stimulation of IP by low [DHO] and inhibition of IP by high [DHO] in 4 and 8 mM [K+]o. In this cell, when [K+]o was 4 mM, the stimulation of IP was 26 pA and the inhibition was 71 pA. When [K+]o was increased to 8 mM, the stimulation of IP was 27 pA and the inhibition was 96 pA. Thus activity of the α1-isoform increased, but the stimulation did not. (B) Average results from five cells. In each cell, the stimulation by 10 nM DHO and the inhibition by 1 mM DHO were recorded in 4 and 8 mM [K+]o, and then the ratio of the stimulation of IP (1.04 ± 0.08, P = 0.31) at the two [K+]o and the ratio of the total IP (1.39 ± 0.20, P = 0.029) at the two [K+]o were recorded and averaged.

Mentions: Gao et al. (1995) showed that the [K+]o affinity of the α2-isoform is much higher (K1/2 = 0.4 mM) than that of the α1-isoform (K1/2 = 4 mM). Thus at 4 mM [K+]o, α2-isoform activity is at 0.92 of saturation, whereas α1-isoform is at half-saturation. Hence, if [K+]o is changed from 4 to 8 mM, α1-isoform activity will increase significantly, whereas α2-isoform activity will increase very little. If the stimulation of IP by nanomolar [DHO] is not via the α1-isoform, then changing [K+]o from 4 to 8 mM will not change the stimulation of IP very much, but it will increase total IP by increasing α1-isoform activity. Therefore, two predictions emerge: (1) in each cell, the ratio of the stimulation of IP in 8 to 4 mM [K+]o will be ∼1.04, given the average numbers presented in Gao et al. (1995); (2) in each cell, the ratio of total IP in 8 to 4 mM [K+]o will be ∼1.4, again based on average numbers presented in Gao et al., 1995. Fig. 3 A shows an example of the protocol. In this cell, at 4 mM [K+]o the stimulation of IP by 10 nM DHO was 26 pA, and the total IP indicated by 1 mM DHO was 71 pA. In the same cell when the external solution contained 8 mM [K+]o, the stimulation of IP was 27 pA and total IP indicated by 1 mM DHO was 96 pA. Thus, the ratio of the stimulation at 8 to 4 mM [K+]o was 1.04, whereas the ratio of total IP in 8 to 4 mM [K+]o was 1.35. Fig. 3 B summarizes the results from a total of five cells. The ratio of IP stimulation in 8 to 4 mM [K+]o was 1.04 ± 0.08, and the ratio of total IP increased in all cells by an average value of 1.39 ± 0.20. These results are consistent with the predictions and further support the hypothesis that stimulation of IP is not associated with the α1-isoform of the Na/K ATPase.


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)

[K+]o modulation of IP in guinea pig ventricular myocytes suggests the α2-isoform and not the α1-isoform is involved in stimulation of IP by low [DHO]. (A) An original record of holding current showing the protocol for observing stimulation of IP by low [DHO] and inhibition of IP by high [DHO] in 4 and 8 mM [K+]o. In this cell, when [K+]o was 4 mM, the stimulation of IP was 26 pA and the inhibition was 71 pA. When [K+]o was increased to 8 mM, the stimulation of IP was 27 pA and the inhibition was 96 pA. Thus activity of the α1-isoform increased, but the stimulation did not. (B) Average results from five cells. In each cell, the stimulation by 10 nM DHO and the inhibition by 1 mM DHO were recorded in 4 and 8 mM [K+]o, and then the ratio of the stimulation of IP (1.04 ± 0.08, P = 0.31) at the two [K+]o and the ratio of the total IP (1.39 ± 0.20, P = 0.029) at the two [K+]o were recorded and averaged.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2238186&req=5

fig5: [K+]o modulation of IP in guinea pig ventricular myocytes suggests the α2-isoform and not the α1-isoform is involved in stimulation of IP by low [DHO]. (A) An original record of holding current showing the protocol for observing stimulation of IP by low [DHO] and inhibition of IP by high [DHO] in 4 and 8 mM [K+]o. In this cell, when [K+]o was 4 mM, the stimulation of IP was 26 pA and the inhibition was 71 pA. When [K+]o was increased to 8 mM, the stimulation of IP was 27 pA and the inhibition was 96 pA. Thus activity of the α1-isoform increased, but the stimulation did not. (B) Average results from five cells. In each cell, the stimulation by 10 nM DHO and the inhibition by 1 mM DHO were recorded in 4 and 8 mM [K+]o, and then the ratio of the stimulation of IP (1.04 ± 0.08, P = 0.31) at the two [K+]o and the ratio of the total IP (1.39 ± 0.20, P = 0.029) at the two [K+]o were recorded and averaged.
Mentions: Gao et al. (1995) showed that the [K+]o affinity of the α2-isoform is much higher (K1/2 = 0.4 mM) than that of the α1-isoform (K1/2 = 4 mM). Thus at 4 mM [K+]o, α2-isoform activity is at 0.92 of saturation, whereas α1-isoform is at half-saturation. Hence, if [K+]o is changed from 4 to 8 mM, α1-isoform activity will increase significantly, whereas α2-isoform activity will increase very little. If the stimulation of IP by nanomolar [DHO] is not via the α1-isoform, then changing [K+]o from 4 to 8 mM will not change the stimulation of IP very much, but it will increase total IP by increasing α1-isoform activity. Therefore, two predictions emerge: (1) in each cell, the ratio of the stimulation of IP in 8 to 4 mM [K+]o will be ∼1.04, given the average numbers presented in Gao et al. (1995); (2) in each cell, the ratio of total IP in 8 to 4 mM [K+]o will be ∼1.4, again based on average numbers presented in Gao et al., 1995. Fig. 3 A shows an example of the protocol. In this cell, at 4 mM [K+]o the stimulation of IP by 10 nM DHO was 26 pA, and the total IP indicated by 1 mM DHO was 71 pA. In the same cell when the external solution contained 8 mM [K+]o, the stimulation of IP was 27 pA and total IP indicated by 1 mM DHO was 96 pA. Thus, the ratio of the stimulation at 8 to 4 mM [K+]o was 1.04, whereas the ratio of total IP in 8 to 4 mM [K+]o was 1.35. Fig. 3 B summarizes the results from a total of five cells. The ratio of IP stimulation in 8 to 4 mM [K+]o was 1.04 ± 0.08, and the ratio of total IP increased in all cells by an average value of 1.39 ± 0.20. These results are consistent with the predictions and further support the hypothesis that stimulation of IP is not associated with the α1-isoform of the Na/K ATPase.

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