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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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ΔIp-[OUA] relation in guinea pig ventricular myocytes. (A) A sample of raw data showing the protocol for observing stimulation of Ip by low [OUA] and inhibition of Ip by high [OUA]. In this cell, four different concentrations of OUA were applied. The three low concentrations of OUA stimulated Ip and 0.5 mM OUA completely inhibited Ip (see B). Dotted line indicates the level of Ip in the absence of OUA. (B) Normalized ΔIp-[OUA] curve. In the absence of OUA, ΔIp was 0 (straight dotted line). The normalized ΔIp at each point was averaged from at least five cells, and the bars indicate SD. The points above the zero level indicate the stimulation of Ip and those points below the zero level represent the inhibition of IP. Ip was maximally stimulated by 1 nM OUA and completely inhibited by 0.5 mM OUA. The smooth curves were obtained by curve fitting the points in the figure with Eq. 1. IP1 + IP2 = IPT. The best-fit parameters were k = 1.36,  = 2.12 × 10−10 M,  = 3 × 10−8 M, K1 = 6.18 × 10−6 M, f2 = 0.24, f1 = 0.76. The positive values of ΔIP at ouabain concentrations of 10−10, 10−9, and 10−8 M represent statistically significant increases in the holding current with P values of 10−3, 2 × 10−4, and 10−2, respectively.
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fig10: ΔIp-[OUA] relation in guinea pig ventricular myocytes. (A) A sample of raw data showing the protocol for observing stimulation of Ip by low [OUA] and inhibition of Ip by high [OUA]. In this cell, four different concentrations of OUA were applied. The three low concentrations of OUA stimulated Ip and 0.5 mM OUA completely inhibited Ip (see B). Dotted line indicates the level of Ip in the absence of OUA. (B) Normalized ΔIp-[OUA] curve. In the absence of OUA, ΔIp was 0 (straight dotted line). The normalized ΔIp at each point was averaged from at least five cells, and the bars indicate SD. The points above the zero level indicate the stimulation of Ip and those points below the zero level represent the inhibition of IP. Ip was maximally stimulated by 1 nM OUA and completely inhibited by 0.5 mM OUA. The smooth curves were obtained by curve fitting the points in the figure with Eq. 1. IP1 + IP2 = IPT. The best-fit parameters were k = 1.36, = 2.12 × 10−10 M, = 3 × 10−8 M, K1 = 6.18 × 10−6 M, f2 = 0.24, f1 = 0.76. The positive values of ΔIP at ouabain concentrations of 10−10, 10−9, and 10−8 M represent statistically significant increases in the holding current with P values of 10−3, 2 × 10−4, and 10−2, respectively.

Mentions: Our studies with DHO, chosen for its rapid binding and unbinding, showed that low concentrations (10−9 to 10−7 M) resulted in an increase in outward current. However, they did not demonstrate whether this “pump stimulation” was a specific action of DHO or a more general property of a class of compounds. We therefore performed similar studies with ouabain (OUA). The results of these studies are provided in Fig. 8 (A and B). A sample of the protocol is provided in Fig. 8 A. An increase in outward current is observed in response to application of 10−10 M OUA. The maximum outward current is observed at 1 nM (a concentration about an order of magnitude lower than the [DHO] required for its maximal stimulatory effect). A smaller outward current shift is observed at 10−8 M OUA. On application of 5 × 10−4 M, there is a large inward shift in holding current corresponding to total inhibition of the Na/K pump. When OUA is washed out, there is a slow return to the original holding current.


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)

ΔIp-[OUA] relation in guinea pig ventricular myocytes. (A) A sample of raw data showing the protocol for observing stimulation of Ip by low [OUA] and inhibition of Ip by high [OUA]. In this cell, four different concentrations of OUA were applied. The three low concentrations of OUA stimulated Ip and 0.5 mM OUA completely inhibited Ip (see B). Dotted line indicates the level of Ip in the absence of OUA. (B) Normalized ΔIp-[OUA] curve. In the absence of OUA, ΔIp was 0 (straight dotted line). The normalized ΔIp at each point was averaged from at least five cells, and the bars indicate SD. The points above the zero level indicate the stimulation of Ip and those points below the zero level represent the inhibition of IP. Ip was maximally stimulated by 1 nM OUA and completely inhibited by 0.5 mM OUA. The smooth curves were obtained by curve fitting the points in the figure with Eq. 1. IP1 + IP2 = IPT. The best-fit parameters were k = 1.36,  = 2.12 × 10−10 M,  = 3 × 10−8 M, K1 = 6.18 × 10−6 M, f2 = 0.24, f1 = 0.76. The positive values of ΔIP at ouabain concentrations of 10−10, 10−9, and 10−8 M represent statistically significant increases in the holding current with P values of 10−3, 2 × 10−4, and 10−2, respectively.
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Related In: Results  -  Collection

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fig10: ΔIp-[OUA] relation in guinea pig ventricular myocytes. (A) A sample of raw data showing the protocol for observing stimulation of Ip by low [OUA] and inhibition of Ip by high [OUA]. In this cell, four different concentrations of OUA were applied. The three low concentrations of OUA stimulated Ip and 0.5 mM OUA completely inhibited Ip (see B). Dotted line indicates the level of Ip in the absence of OUA. (B) Normalized ΔIp-[OUA] curve. In the absence of OUA, ΔIp was 0 (straight dotted line). The normalized ΔIp at each point was averaged from at least five cells, and the bars indicate SD. The points above the zero level indicate the stimulation of Ip and those points below the zero level represent the inhibition of IP. Ip was maximally stimulated by 1 nM OUA and completely inhibited by 0.5 mM OUA. The smooth curves were obtained by curve fitting the points in the figure with Eq. 1. IP1 + IP2 = IPT. The best-fit parameters were k = 1.36, = 2.12 × 10−10 M, = 3 × 10−8 M, K1 = 6.18 × 10−6 M, f2 = 0.24, f1 = 0.76. The positive values of ΔIP at ouabain concentrations of 10−10, 10−9, and 10−8 M represent statistically significant increases in the holding current with P values of 10−3, 2 × 10−4, and 10−2, respectively.
Mentions: Our studies with DHO, chosen for its rapid binding and unbinding, showed that low concentrations (10−9 to 10−7 M) resulted in an increase in outward current. However, they did not demonstrate whether this “pump stimulation” was a specific action of DHO or a more general property of a class of compounds. We therefore performed similar studies with ouabain (OUA). The results of these studies are provided in Fig. 8 (A and B). A sample of the protocol is provided in Fig. 8 A. An increase in outward current is observed in response to application of 10−10 M OUA. The maximum outward current is observed at 1 nM (a concentration about an order of magnitude lower than the [DHO] required for its maximal stimulatory effect). A smaller outward current shift is observed at 10−8 M OUA. On application of 5 × 10−4 M, there is a large inward shift in holding current corresponding to total inhibition of the Na/K pump. When OUA is washed out, there is a slow return to the original holding current.

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
Related in: MedlinePlus