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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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The effects of DHO on Na/K pump current in human heart cells. Stimulation of IP by low [DHO] and inhibition of IP by high [DHO] were observed in human ventricular myocytes (A) and in human atrial myocytes (B). (C) The effects of β-adrenergic activation (ISO) and α-adrenergic activation (NE + PROP) on IP in human atrial cells. Bars indicate standard deviations. The numbers in the parentheses represent the number of cells studied. There was no significant effect of ISO (P = 0.50). However, there was a significant difference between IP in control and that in the presence of α-adrenergic activation (P = 0.009). (D) The ΔIP-DHO curve in human atrial cells. ΔIP was normalized as described in Fig. 6 A. Data were fitted assuming the presence of only one DHO affinity pump, since the α1-isoform of the Na/K pump in human heart has essentially the same DHO affinity as the α2- and α3-isoforms. The positive values of ΔIP at DHO concentrations of 10−9, 10−8, and 10−7 M represent statistically significant increases in the holding current with P values of 3 × 10−3, 10−3, and 4 × 10−3, respectively.
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fig12: The effects of DHO on Na/K pump current in human heart cells. Stimulation of IP by low [DHO] and inhibition of IP by high [DHO] were observed in human ventricular myocytes (A) and in human atrial myocytes (B). (C) The effects of β-adrenergic activation (ISO) and α-adrenergic activation (NE + PROP) on IP in human atrial cells. Bars indicate standard deviations. The numbers in the parentheses represent the number of cells studied. There was no significant effect of ISO (P = 0.50). However, there was a significant difference between IP in control and that in the presence of α-adrenergic activation (P = 0.009). (D) The ΔIP-DHO curve in human atrial cells. ΔIP was normalized as described in Fig. 6 A. Data were fitted assuming the presence of only one DHO affinity pump, since the α1-isoform of the Na/K pump in human heart has essentially the same DHO affinity as the α2- and α3-isoforms. The positive values of ΔIP at DHO concentrations of 10−9, 10−8, and 10−7 M represent statistically significant increases in the holding current with P values of 3 × 10−3, 10−3, and 4 × 10−3, respectively.

Mentions: As described in the introduction, guinea pig ventricular myocytes coexpress the α1- and α2-isoforms of the Na/K pump (Gao et al., 1995). Since these two isoforms have an ∼100-fold difference in affinity for the cardiac glycosides, they can be studied separately by using 5 μM DHO to block the current generated by only the α2-isoform (IP2), and using 1 mM DHO to block total current (IP) generated by the both isoforms. Total pump current is given by IP = IP1 + IP2. With physiological pH and [K+]o, IP1 is ∼60% of IP, but there is some cell to cell variation in this percent. There also is considerable cell to cell variation in myocyte size as well as in the density of total pumps per square centimeter of cell membrane. The original records displayed in this section reflect the variability in all of these parameters, so a wide range of current scales are used to optimally display each individual record. However, when we studied an effector of pump current, the standard protocol was to measure pump current in control, test conditions in the same cell, and then calculate the ratio of test to control current and average this ratio from at least five cells. This procedure uses each cell as its own control and removes the uncertainty due to cell to cell variation in size and pump density. In the data that follow, when we report an effect on the pump current, that effect was relative to control conditions in the same cell, and each effect was observed in 100% of the cells in which the protocol was completed. This procedure requires that each cell be held stably in the whole-cell patch configuration for time periods in excess of 10 min. The human heart cell data shown in Fig. 10 C are the only exception to the above protocol. After patch clamping these cells, they generally survived only a few minutes. Therefore, we made a quick measurement of cell capacitance at the beginning of each experiment, and this was used as a measure of cell size to normalize the subsequent measurements 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)

The effects of DHO on Na/K pump current in human heart cells. Stimulation of IP by low [DHO] and inhibition of IP by high [DHO] were observed in human ventricular myocytes (A) and in human atrial myocytes (B). (C) The effects of β-adrenergic activation (ISO) and α-adrenergic activation (NE + PROP) on IP in human atrial cells. Bars indicate standard deviations. The numbers in the parentheses represent the number of cells studied. There was no significant effect of ISO (P = 0.50). However, there was a significant difference between IP in control and that in the presence of α-adrenergic activation (P = 0.009). (D) The ΔIP-DHO curve in human atrial cells. ΔIP was normalized as described in Fig. 6 A. Data were fitted assuming the presence of only one DHO affinity pump, since the α1-isoform of the Na/K pump in human heart has essentially the same DHO affinity as the α2- and α3-isoforms. The positive values of ΔIP at DHO concentrations of 10−9, 10−8, and 10−7 M represent statistically significant increases in the holding current with P values of 3 × 10−3, 10−3, and 4 × 10−3, respectively.
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Related In: Results  -  Collection

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fig12: The effects of DHO on Na/K pump current in human heart cells. Stimulation of IP by low [DHO] and inhibition of IP by high [DHO] were observed in human ventricular myocytes (A) and in human atrial myocytes (B). (C) The effects of β-adrenergic activation (ISO) and α-adrenergic activation (NE + PROP) on IP in human atrial cells. Bars indicate standard deviations. The numbers in the parentheses represent the number of cells studied. There was no significant effect of ISO (P = 0.50). However, there was a significant difference between IP in control and that in the presence of α-adrenergic activation (P = 0.009). (D) The ΔIP-DHO curve in human atrial cells. ΔIP was normalized as described in Fig. 6 A. Data were fitted assuming the presence of only one DHO affinity pump, since the α1-isoform of the Na/K pump in human heart has essentially the same DHO affinity as the α2- and α3-isoforms. The positive values of ΔIP at DHO concentrations of 10−9, 10−8, and 10−7 M represent statistically significant increases in the holding current with P values of 3 × 10−3, 10−3, and 4 × 10−3, respectively.
Mentions: As described in the introduction, guinea pig ventricular myocytes coexpress the α1- and α2-isoforms of the Na/K pump (Gao et al., 1995). Since these two isoforms have an ∼100-fold difference in affinity for the cardiac glycosides, they can be studied separately by using 5 μM DHO to block the current generated by only the α2-isoform (IP2), and using 1 mM DHO to block total current (IP) generated by the both isoforms. Total pump current is given by IP = IP1 + IP2. With physiological pH and [K+]o, IP1 is ∼60% of IP, but there is some cell to cell variation in this percent. There also is considerable cell to cell variation in myocyte size as well as in the density of total pumps per square centimeter of cell membrane. The original records displayed in this section reflect the variability in all of these parameters, so a wide range of current scales are used to optimally display each individual record. However, when we studied an effector of pump current, the standard protocol was to measure pump current in control, test conditions in the same cell, and then calculate the ratio of test to control current and average this ratio from at least five cells. This procedure uses each cell as its own control and removes the uncertainty due to cell to cell variation in size and pump density. In the data that follow, when we report an effect on the pump current, that effect was relative to control conditions in the same cell, and each effect was observed in 100% of the cells in which the protocol was completed. This procedure requires that each cell be held stably in the whole-cell patch configuration for time periods in excess of 10 min. The human heart cell data shown in Fig. 10 C are the only exception to the above protocol. After patch clamping these cells, they generally survived only a few minutes. Therefore, we made a quick measurement of cell capacitance at the beginning of each experiment, and this was used as a measure of cell size to normalize the subsequent measurements 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