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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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Adrenergic 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) A typical record of holding current in an experiment showing the effect of β-adrenergic activation with ISO on stimulation of IP by 10 nM DHO. The vertical bar labeled with ΔIP indicates the magnitude of the stimulation of IP. The large increase in holding current in the presence of ISO is mainly due to the activation of the Cl− conductance (Harvey and Hume, 1989; Bahinski et al., 1989). (B) Summary of the results from a total of five cells. ΔIP was normalized by its value in control conditions. The normalized ΔIP in the presence of ISO was 0.98 ± 0.04 (SD), suggesting that ISO had no effect on the stimulation (P = 0.51). (C) A typical record of holding current in an experiment showing the effect of α-adrenergic activation (NE and PROP) on ΔIP. (D) Summary of the results from a total of five cells. The normalized ΔIP in the presence of α-activation was 1.54 ± 0.11 (SD), suggesting that α activation enhanced the stimulation of IP (P = 0.008).
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fig4: Adrenergic 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) A typical record of holding current in an experiment showing the effect of β-adrenergic activation with ISO on stimulation of IP by 10 nM DHO. The vertical bar labeled with ΔIP indicates the magnitude of the stimulation of IP. The large increase in holding current in the presence of ISO is mainly due to the activation of the Cl− conductance (Harvey and Hume, 1989; Bahinski et al., 1989). (B) Summary of the results from a total of five cells. ΔIP was normalized by its value in control conditions. The normalized ΔIP in the presence of ISO was 0.98 ± 0.04 (SD), suggesting that ISO had no effect on the stimulation (P = 0.51). (C) A typical record of holding current in an experiment showing the effect of α-adrenergic activation (NE and PROP) on ΔIP. (D) Summary of the results from a total of five cells. The normalized ΔIP in the presence of α-activation was 1.54 ± 0.11 (SD), suggesting that α activation enhanced the stimulation of IP (P = 0.008).

Mentions: We have shown previously that β-adrenergic activation, through activation of PKA, specifically increases the current generated by the α1-isoform (for review see Mathias et al., 2000), either by increasing the number of pumps in the plasma membrane or by increasing the turnover rate of each pump. In either situation, if the stimulation of IP by nanomolar [DHO] is via the α1-isoform, the stimulation should increase in the presence of β-adrenergic activation. We examined the effects of β-adrenergic activation with the specific β-agonist isoproterenol (ISO). Fig. 2 A shows the effect of ISO on the stimulation of IP by 10 nM DHO. In this example, stimulation of IP in the control solution (IP(Con)) and that in the presence of ISO (IP(ISO)) are 11 and 12 pA, respectively. The summary of the results from a total of five cells is shown in Fig. 2 B. The stimulation of IP(Con) was normalized to 1. Then, the ratio IP(ISO)/IP(Con) in each cell was averaged to obtain the value 0.98 ± 0.04 (SD), indicating ISO had no effect on the stimulation of IP, suggesting the α1-isoform is not involved.


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)

Adrenergic 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) A typical record of holding current in an experiment showing the effect of β-adrenergic activation with ISO on stimulation of IP by 10 nM DHO. The vertical bar labeled with ΔIP indicates the magnitude of the stimulation of IP. The large increase in holding current in the presence of ISO is mainly due to the activation of the Cl− conductance (Harvey and Hume, 1989; Bahinski et al., 1989). (B) Summary of the results from a total of five cells. ΔIP was normalized by its value in control conditions. The normalized ΔIP in the presence of ISO was 0.98 ± 0.04 (SD), suggesting that ISO had no effect on the stimulation (P = 0.51). (C) A typical record of holding current in an experiment showing the effect of α-adrenergic activation (NE and PROP) on ΔIP. (D) Summary of the results from a total of five cells. The normalized ΔIP in the presence of α-activation was 1.54 ± 0.11 (SD), suggesting that α activation enhanced the stimulation of IP (P = 0.008).
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Adrenergic 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) A typical record of holding current in an experiment showing the effect of β-adrenergic activation with ISO on stimulation of IP by 10 nM DHO. The vertical bar labeled with ΔIP indicates the magnitude of the stimulation of IP. The large increase in holding current in the presence of ISO is mainly due to the activation of the Cl− conductance (Harvey and Hume, 1989; Bahinski et al., 1989). (B) Summary of the results from a total of five cells. ΔIP was normalized by its value in control conditions. The normalized ΔIP in the presence of ISO was 0.98 ± 0.04 (SD), suggesting that ISO had no effect on the stimulation (P = 0.51). (C) A typical record of holding current in an experiment showing the effect of α-adrenergic activation (NE and PROP) on ΔIP. (D) Summary of the results from a total of five cells. The normalized ΔIP in the presence of α-activation was 1.54 ± 0.11 (SD), suggesting that α activation enhanced the stimulation of IP (P = 0.008).
Mentions: We have shown previously that β-adrenergic activation, through activation of PKA, specifically increases the current generated by the α1-isoform (for review see Mathias et al., 2000), either by increasing the number of pumps in the plasma membrane or by increasing the turnover rate of each pump. In either situation, if the stimulation of IP by nanomolar [DHO] is via the α1-isoform, the stimulation should increase in the presence of β-adrenergic activation. We examined the effects of β-adrenergic activation with the specific β-agonist isoproterenol (ISO). Fig. 2 A shows the effect of ISO on the stimulation of IP by 10 nM DHO. In this example, stimulation of IP in the control solution (IP(Con)) and that in the presence of ISO (IP(ISO)) are 11 and 12 pA, respectively. The summary of the results from a total of five cells is shown in Fig. 2 B. The stimulation of IP(Con) was normalized to 1. Then, the ratio IP(ISO)/IP(Con) in each cell was averaged to obtain the value 0.98 ± 0.04 (SD), indicating ISO had no effect on the stimulation of IP, suggesting the α1-isoform is not involved.

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