scholarly journals Isoform-specific Stimulation of Cardiac Na/K Pumps by Nanomolar Concentrations of Glycosides

2002 ◽  
Vol 119 (4) ◽  
pp. 297-312 ◽  
Author(s):  
Junyuan Gao ◽  
Randy S. Wymore ◽  
Yongli Wang ◽  
Glenn R. Gaudette ◽  
Irvin B. Krukenkamp ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Outward Current ◽  
Pump Current ◽  
Ion Accumulation ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Pump Activity ◽  
Specific Regulation ◽  
Stimulation Of

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 (IP) 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 IP because of the following: (1) it was absent in 0 mM [K+]o, as was IP; (2) it was absent in 0 mM [Na+]i, as was IP; (3) at reduced [Na+]i, the outward current was reduced in proportion to the reduction in IP; (4) it was eliminated by intracellular vanadate, as was IP. Our previous work suggested guinea pig ventricular myocytes coexpress the α1- and α2-isoforms of the Na/K pumps. The stimulation of IP appears to be through stimulation of the high glycoside affinity α2-isoform and not the α1-isoform because of the following: (1) regulatory signals that specifically increased activity of the α2-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the α1-isoform did not affect the stimulation; (3) changes in [K+]o that affected activity of the α1-isoform, but not the α2-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the α1-isoform but not the α2-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total IP increased by 35 ± 10% (mean ± SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the α2-isoform, then activity of the α2-isoform increased by 107 ± 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the α2-isoform, but both the stimulatory and inhibitory concentrations of ouabain were ∼10-fold lower than those for DHO. Stimulation of IP by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the α1- and α3-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the α3-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 IP that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of IP, and where the contributions of the high glycoside affinity α2- and α3-isoforms could be separated from that of the α1-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 IP in heart by nanomolar concentrations of endogenous ouabain-like molecules.

Alpha 1b-adrenoceptor-mediated stimulation of Na-K pump current in adult rat ventricular myocytes

1993 ◽  
Vol 264 (4) ◽  
pp. H1315-H1318 ◽  
Author(s):  
A. P. Williamson ◽  
R. H. Kennedy ◽  
E. Seifen ◽  
J. P. Lindemann ◽  
J. R. Stimers
Keyword(s):  
Adrenergic Receptors ◽  
Ventricular Myocytes ◽  
Pump Current ◽  
Peak Effect ◽  
Patch Clamp Technique ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Whole Cell Patch Clamp ◽  
Dose Dependent ◽  
Stimulation Of

The purpose of this study was to determine if myocardial alpha 1a-and/or alpha 1b-adrenoceptors are involved in the increase in Na-K pump current (Ip) elicited by alpha 1-adrenergic agonists. Single rat ventricular myocytes were isolated by enzymatic disaggregation. The whole cell patch-clamp technique was used to examine dose-dependent effects of phenylephrine (PE) on holding current (Ih) and to determine whether observed actions were mediated via alpha 1a-or alpha 1b-adrenergic receptors. To minimize the contribution of transsar-colemmal currents other than Ip to Ih, membrane voltage was held constant -40 mV, and cells were maintained in a Ca-free perfusate containing 1 mM Ba and 0.1 mM Cd. All experiments were conducted in the presence of 3 microM nadolol. PE elicited dose-dependent increases in Ih, with a peak effect of 0.57 +/- 0.03 pA/pF observed at 30 microM. The response to PE was dose dependently inhibited by prazosin and chloroethylclonidine and was totally eliminated by 1 mM ouabain. When used at doses selective for the alpha 1a-subtype, WB4101 failed to significantly antagonize the action of PE. These data suggest that the observed alpha 1-adrenoceptor-mediated increase in Ih in isolated rat ventricular myocytes is the result of an increase in Ip effected via stimulation of alpha 1b-adrenergic receptors.

Permeability of time-dependent K+ channel in guinea pig ventricular myocytes to Cs+, Na+, NH4+, and Rb+

1990 ◽  
Vol 259 (5) ◽  
pp. H1448-H1454 ◽  
Author(s):  
R. W. Hadley ◽  
J. R. Hume
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Outward Current ◽  
Time Dependent ◽  
K Channel ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Outward Currents

Currents through time-dependent K+ channels (also referred to as IK or the delayed rectifier) were studied with the whole cell patch-clamp technique in isolated guinea pig ventricular myocytes. IK measurements were restricted to the examination of deactivation tail currents. Substitution of various monovalent cations for external K+ produced shifts of the reversal potential of IK. These shifts were used to calculate permeability ratios relative to K+. The permeability sequence for the IK channels was K+ = Rb+ greater than NH4+ = Cs+ greater than Na+. Time-dependent outward currents were also examined when the myocytes were dialyzed with Cs+ instead of K+. A sizeable time-dependent outward current, quite similar to that seen with K+ dialysis, was demonstrated. This current was primarily carried by intracellular Cs+, as the reversal potential of the current shifted 46 mV per 10-fold change of external Cs+ concentration. The significance of Cs+ permeation through IK channels is discussed with respect to the common use of Cs+ in isolating other currents.

Force measurements from voltage-clamped guinea pig ventricular myocytes

1990 ◽  
Vol 258 (2) ◽  
pp. H452-H459 ◽  
Author(s):  
N. Shepherd ◽  
M. Vornanen ◽  
G. Isenberg
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Ventricular Myocytes ◽  
Isometric Force ◽  
Contractile Force ◽  
Patch Clamp Technique ◽  
Thin Glass ◽  
Tonic Component ◽  
Time To Peak ◽  
Whole Cell Patch Clamp

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

PKC regulation of cardiac CFTR Cl− channel function in guinea pig ventricular myocytes

1998 ◽  
Vol 275 (1) ◽  
pp. C293-C302 ◽  
Author(s):  
Lisa M. Middleton ◽  
Robert D. Harvey
Keyword(s):  
Protein Kinase ◽  
Patch Clamp ◽  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Perforated Patch Clamp

The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl− current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl− current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the β-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl− conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% ( n = 9) when added after isoproterenol and by 36 ± 16% ( n= 14) when introduced before isoproterenol. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl− current by 79 ± 7.7% ( n = 11) and 52 ± 10% ( n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl− channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.

Delayed-rectifier potassium channel activity in isolated membrane patches of guinea pig ventricular myocytes

1991 ◽  
Vol 260 (4) ◽  
pp. H1390-H1393 ◽  
Author(s):  
K. B. Walsh ◽  
J. P. Arena ◽  
W. M. Kwok ◽  
L. Freeman ◽  
R. S. Kass
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Outward Current ◽  
Time Dependent ◽  
Delayed Rectifier ◽  
Potential Range ◽  
Ammonium Compound ◽  
Channel Activity ◽  
Patch Clamp Technique

When the patch-clamp technique was used, a slowly activating, time-dependent outward current was identified in both cell-attached and excised membrane patches obtained from guinea pig ventricular myocytes. This macroscopic patch current was present in approximately 50% of patches studied and could be observed both in the presence and absence of unitary single channel activity (i.e., ATP-sensitive K+ channels). The time course of activation of the patch current resembled that of the whole cell delayed-rectifier K+ current (IK) recorded under similar ionic conditions, and the patch current and IK were activated over a similar membrane potential range. The time-dependent patch current could be eliminated when the Nernst potential for K+ equaled that of the pulse voltage. The patch current was inhibited by external addition of the tertiary ammonium compound LY 97241 (50 microM) and was augmented after internal application of the catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (500 nM). Deactivating tail currents with kinetics similar to those of IK could be recorded to cell-attached and excised patches. Unitary single channel events underlying the time-dependent patch current could not be resolved despite various attempts to increase single channel conductance. Thus our results suggest that a major component of delayed rectification in guinea pig ventricular cells is due to the activity of a high-density, extremely low conductance K+ channel.

Divalent cations modulate the transient outward current in rat ventricular myocytes

1991 ◽  
Vol 261 (2) ◽  
pp. C310-C318 ◽  
Author(s):  
Z. S. Agus ◽  
I. D. Dukes ◽  
M. Morad
Keyword(s):  
Hippocampal Neurons ◽  
Voltage Dependence ◽  
Ventricular Myocytes ◽  
Divalent Cations ◽  
Outward Current ◽  
Transient Outward Current ◽  
Patch Clamp Technique ◽  
Rat Ventricular Myocytes ◽  
Specific Effects ◽  
Whole Cell Patch Clamp

The modulation of the transient outward K+ current (Ito) by divalent cations was studied in enzymatically isolated rat ventricular myocytes with the whole cell patch-clamp technique. At holding potentials negative to -70 mV, 1 mM Cd2+ suppressed Ito, whereas, at potentials positive to -50 mV, the current was augmented. These effects were caused by shifts in the voltage dependence of both activation and inactivation of Ito toward more positive potentials. Cd2+ also slowed the activation kinetics of Ito by shifting the voltage dependence of its rate of activation, but the rate of inactivation was unaffected. Other divalent cations produced similar shifts but at markedly different concentrations. Thus, in the millimolar range, a rightward shift of approximately 20 mV was produced by 3 Co2+, 5 Ni2+, and 10 Ca2+, whereas 10 microM concentrations of Cu2+ and Zn2+ produced equivalent shifts. Similar effects were seen in hippocampal neurons with micromolar concentrations of Zn2+. Thus divalent cations have marked and specific effects on the kinetics and voltage dependence of Ito and may serve as a regulatory mechanism in its activation, particularly in cells with resting potentials positive to -60 mV.

Voltage-dependent stimulation of the Na+-K+ pump by insulin in rabbit cardiac myocytes

2000 ◽  
Vol 278 (3) ◽  
pp. C546-C553 ◽  
Author(s):  
Peter S. Hansen ◽  
Kerrie A. Buhagiar ◽  
David F. Gray ◽  
Helge H. Rasmussen
Keyword(s):  
Protein Phosphatase ◽  
Ventricular Myocytes ◽  
Pump Current ◽  
Phosphatidylinositol 3 Kinase ◽  
Patch Clamp Technique ◽  
Membrane Pump ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Phosphatase 2A

Insulin enhances Na+-K+ pump activity in various noncardiac tissues. We examined whether insulin exposure in vitro regulates Na+-K+ pump function in rabbit ventricular myocytes. Pump current ( I p) was measured using the whole-cell patch-clamp technique at test potentials ( V ms) from −100 to +60 mV. When the Na+ concentration in the patch pipette ([Na]pip) was 10 mM, insulin caused a V m-dependent increase in I p. The increase was ∼70% when V m was at near physiological diastolic potentials. This effect persisted after elimination of extracellular voltage-dependent steps and when K+ and K+-congeners were excluded from the patch pipettes. When [Na]pip was 80 mM, causing near-maximal pump stimulation, insulin had no effect, suggesting that it did not cause an increase in membrane pump density. Effects of tyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I in pipette solutions suggested that the insulin-induced increase in I p involved activation of tyrosine kinase, phosphatidylinositol 3-kinase, and protein phosphatase 1, whereas protein phosphatase 2A and protein kinase C were not involved.

Opposing effects of coupled and uncoupled NOS activity on the Na+-K+ pump in cardiac myocytes

2008 ◽  
Vol 294 (2) ◽  
pp. C572-C578 ◽  
Author(s):  
C. N. White ◽  
E. J. Hamilton ◽  
A. Garcia ◽  
D. Wang ◽  
K. K. M. Chia ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Superoxide Dismutase ◽  
Cardiac Myocytes ◽  
Pump Current ◽  
No Synthase ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Patch Pipette ◽  
Pump Activity ◽  
Opposing Effects

Pharmacological delivery of nitric oxide (NO) stimulates the cardiac Na+-K+ pump. However, effects of NO synthesized by NO synthase (NOS) often differ from the effects of NO delivered pharmacologically. In addition, NOS can become “uncoupled” and preferentially synthesize O2·−, which often has opposing effects to NO. We tested the hypothesis that NOS-synthesized NO stimulates Na+-K+ pump activity, and uncoupling of NOS inhibits it. To image NO, we loaded isolated rabbit cardiac myocytes with 4,5-diaminofluorescein-2 diacetate (DAF-2 DA) and measured fluorescence with confocal microscopy. l-Arginine (l-Arg; 500 μmol/l) increased DAF-2 DA fluorescence by 51% compared with control ( n = 8; P < 0.05). We used the whole cell patch-clamp technique to measure electrogenic Na+-K+ pump current ( Ip). Mean Ip of 0.35 ± 0.03 pA/pF ( n = 44) was increased to 0.48 ± 0.03 pA/pF ( n = 7, P < 0.05) by 10 μmol/l l-Arg in pipette solutions. This increase was abolished by NOS inhibition with radicicol or by NO-activated guanylyl cyclase inhibition with 1 H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. We next examined the effect of uncoupling NOS using paraquat. Paraquat (1 mmol/l) induced a 51% increase in the fluorescence intensity of O2·−-sensitive dye dihydroethidium compared with control ( n = 9; P < 0.05). To examine the functional effects of uncoupling, we measured Ip with 100 μmol/l paraquat included in patch pipette solutions. This decreased Ip to 0.28 ± 0.03 pA/pF ( n = 12; P < 0.001). The paraquat-induced pump inhibition was abolished by superoxide dismutase (in pipette solutions). We conclude that NOS-mediated NO synthesis stimulates the Na+-K+ pump, whereas uncoupling of NOS causes O2·−-mediated pump inhibition.

Monensin-Induced Increase in Intracellular Na+ Induces Changes in Na+ and Ca2+ Currents and Regulates Na+-K+ and Na+-Ca2+ Transport in Cardiomyocytes

Pharmacology ◽  
2020 ◽  
pp. 1-15
Author(s):  
Katsuharu Tsuchida ◽  
Hitomi Hirose ◽  
Sachiyo Ozawa ◽  
Haruka Ishida ◽  
Tomomi Iwatani ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Sodium Current ◽  
Pump Current ◽  
Plateau Phase ◽  
Patch Clamp Technique ◽  
Inactivation Curve ◽  
Reverse Mode ◽  
Whole Cell Patch Clamp ◽  
Intracellular Ca ◽  
K Current

<b><i>Background/Aims:</i></b> Monensin, an Na ionophore, increases intracellular Na ([Na]i). Alteration of [Na]i influences ion transport through the sarcolemmal membrane. So far, the effects of monensin on ventricular myocytes have not been examined in detail. The main objective of this study was to elucidate the mechanism via which monensin-evoked increases in [Na]i affect the membrane potential and currents in ventricular myocytes of guinea pigs. Methods: Membrane potentials and currents were measured using the whole-cell patch-clamp technique in single myocytes. The concentration of intracellular Ca ([Ca]i) was evaluated by measuring fluorescence intensity of Fluo-4. Results: Monensin (10<sup>−5</sup>M) shortened the action potential duration (APD) and reduced the amplitude of the plateau phase. In addition, monensin decreased the sodium current (I<sub>Na</sub>) and shifted the inactivation curve to the hyperpolarized direction. Moreover, it decreased the L-type calcium current (I<sub>Ca</sub>). However, this effect was attenuated by increasing the buffering capacity of [Ca]i. The Na-Ca exchange current (I<sub>Na-Ca</sub>) was activated particularly in the reverse mode. Na-K pump current (I<sub>Na-K</sub>) was also activated. Notably, the inward rectifying K current (I<sub>K1</sub>) was not affected, and the change in the delayed outward K current (I<sub>K</sub>) was not evident. Conclusion: These results suggest that the monensin-induced shortened APD and reduced amplitude of the plateau phase are primarily due to the decrease in the I<sub>Ca</sub>, the activation of the reverse mode of I<sub>Na-Ca</sub>, and the increased I<sub>Na-K</sub>, and second due to the decreased I<sub>Na</sub>. The I<sub>K</sub> and the I<sub>K1</sub> may not be associated with the abovementioned changes induced by monensin. The elevation of [Na]i can exert multiple influences on electrophysiological phenomena in cardiac myocytes.

scholarly journals Two functionally different Na/K pumps in cardiac ventricular myocytes.

1995 ◽  
Vol 106 (5) ◽  
pp. 995-1030 ◽  
Author(s):  
J Gao ◽  
R T Mathias ◽  
I S Cohen ◽  
G J Baldo
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Dissociation Constants ◽  
Pump Current ◽  
Extracellular Ph ◽  
Patch Clamp Technique ◽  
Maximal Activation ◽  
Different Types ◽  
Physiological Variations

The whole-cell patch-clamp technique was used to voltage clamp acutely isolated myocytes at -60 mV and study effects of ionic environment on Na/K pump activity. In quiescent guinea pig myocytes, normal intracellular Na+ is approximately 6 mM, which gives a total pump current of 0.25 +/- 0.09 pA/pF, and an inward background sodium current of 0.75 +/- 0.26 pA/pF. The average capacitance of a cell is 189 +/- 61 pF. Our main conclusion is the total Na/K pump current comprises currents from two different types of pumps, whose functional responses to the extracellular environment are different. Pump current was reversibly blocked with two affinities by extracellular dihydro-ouabain (DHO). We determined dissociation constants of 72 microM for low affinity (type-1) pumps and 0.75 microM for high affinity (type-h) pumps. These dissociation constants did not detectably change with two intracellular Na+ concentrations, one saturating and one near half-saturating, and with two extracellular K+ concentrations of 4.6 and 1.0 mM. Ion effects on type-h pumps were therefore measured using 5 microM DHO and on total pump current using 1 mM DHO. Extracellular K+ half-maximally activated the type-h pumps at 0.4 mM and the type-1 at 3.7 mM. Extracellular H+ blocked the type-1 pumps with half-maximal blockade at a pH of 7.71 whereas the type-h pumps were insensitive to extracellular pH. Both types of pumps responded similarly to changes in intracellular-Na+, with 9.6 mM causing half-maximal activation. Neither changes in intracellular pH between 6.0 and 7.2, nor concentrations of intracellular K+ of 140 mM or below, had any effect on either type of pump. The lack of any effect of intracellular K+ suggests the dissociation constants are in the molar range so this step in the pump cycle is not rate limiting under normal physiological conditions. Changes in intracellular-Na+ did not affect the half-maximal activation by extracellular K+, and vice versa. We found DHO-blockade of Na/K pump current in canine ventricular myocytes also occurred with two affinities, which are very similar to those from guinea pig myocytes or rat ventricular myocytes. In contrast, isolated canine Purkinje myocytes have predominantly the type-h pumps, insofar as DHO-blockade and extracellular K+ activation are much closer to our type-h results than type-1. These observations suggest for mammalian ventricular myocytes: (a) the presence of two types of Na/K pumps may be a general property. (b) Normal physiological variations in extracellular pH and K+ are important determinants of Na/K pump current. (c) Normal physiological variations in the intracellular environment affect Na/K pump current primarily via the Na+ concentration. Lastly, Na/K pump current appears to be specifically tailored for a tissue by expression of a mix of functionally different types of pumps.

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