Alloxan-induced diabetes reduces sarcolemmal Na+-K+ pump function in rabbit ventricular myocytes

2007 ◽  
Vol 292 (3) ◽  
pp. C1070-C1077 ◽  
Author(s):  
Peter S. Hansen ◽  
Ronald J. Clarke ◽  
Kerrie A. Buhagiar ◽  
Elisha Hamilton ◽  
Alvaro Garcia ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Pump Current ◽  
Pharmacological Intervention ◽  
Angiotensin Ii Receptor ◽  
Metabolic Disturbances ◽  
Patch Clamp Technique ◽  
Pump Function ◽  
Whole Cell Patch Clamp ◽  
Diabetic Rabbits

The effect of diabetes on sarcolemmal Na+-K+ pump function is important for our understanding of heart disease associated with diabetes and design of its treatment. We induced diabetes characterized by hyperglycemia but no other major metabolic disturbances in rabbits. Ventricular myocytes isolated from diabetic rabbits and controls were voltage clamped and internally perfused with the whole cell patch-clamp technique. Electrogenic Na+-K+ pump current ( Ip, arising from the 3:2 Na+-to-K+ exchange ratio) was identified as the shift in holding current induced by Na+-K+ pump blockade with 100 μmol/l ouabain in most experiments. There was no effect of diabetes on Ip recorded when myocytes were perfused with pipette solutions containing 80 mmol/l Na+ to nearly saturate intracellular Na+-K+ pump sites. However, diabetes was associated with a significant decrease in Ip measured when pipette solutions contained 10 mmol/l Na+. The decrease was independent of membrane voltage but dependent on the intracellular concentration of K+. There was no effect of diabetes on the sensitivity of Ip to extracellular K+. Pump inhibition was abolished by restoration of euglycemia or by in vivo angiotensin II receptor blockade with losartan. We conclude that diabetes induces sarcolemmal Na+-K+ pump inhibition that can be reversed with pharmacological intervention.

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.

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.

Dietary cholesterol affects Na+-K+ pump function in rabbit cardiac myocytes

1997 ◽  
Vol 272 (4) ◽  
pp. H1680-H1689 ◽  
Author(s):  
D. F. Gray ◽  
P. S. Hansen ◽  
M. M. Doohan ◽  
L. C. Hool ◽  
H. H. Rasmussen
Keyword(s):  
Cardiac Myocytes ◽  
Serum Cholesterol ◽  
Dietary Cholesterol ◽  
Membrane Cholesterol ◽  
Patch Clamp Technique ◽  
Apparent Affinity ◽  
Pump Function ◽  
Whole Cell Patch Clamp

Alterations in membrane cholesterol induced in vitro can alter Na+-K+ pump function. Because dietary cholesterol can influence membrane cholesterol in vivo, we examined if dietary cholesterol is a determinant of Na+-K+ pump function. Rabbits were fed cholesterol-supplemented diets for 1-4 wk. Cardiac myocytes were then isolated, and Na+-K+ pump currents (Ip) were measured using the whole cell patch-clamp technique. When the Na+ concentration in the patch pipettes ([Na]pip) was 10 mM, a modest diet-induced increase in serum cholesterol was associated with stimulation of Ip; large increases in serum cholesterol were associated with inhibition. There was no effect of modest or large increases in serum cholesterol on Ip when [Na]pip was 80 mM. The [Na]pip-Ip relationship determined using seven different levels of [Na]pip from 0 to 80 mM indicated that a modest increase in serum cholesterol increased the apparent affinity of the pump for cytoplasmic Na+. In contrast, dietary cholesterol had no effect on the apparent affinity of the pump for extracellular K+. We conclude that cholesterol intake influences the sarcolemmal Na+-K+ pump. This may have clinical implications for cardiovascular function.

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.

Mechanisms underlying delayed afterdepolarizations in hypertrophied left ventricular myocytes of rats

2001 ◽  
Vol 281 (2) ◽  
pp. H903-H914 ◽  
Author(s):  
János Mészáros ◽  
Daniel Khananshvili ◽  
George Hart
Keyword(s):  
Ventricular Myocytes ◽  
Pump Current ◽  
Left Ventricular ◽  
Daily Injection ◽  
Nonselective Cation Channel ◽  
Patch Clamp Technique ◽  
Inward Current ◽  
Na Pump ◽  
Whole Cell Patch Clamp ◽  
Delayed Afterdepolarizations

Cardiac hypertrophy was induced in rats by daily injection of isoproterenol (5 mg/kg ip) for 7 days. Membrane voltage and currents were recorded using the whole cell patch-clamp technique in left ventricular myocytes from control and hypertrophied hearts. Ryanodine-sensitive delayed afterdepolarizations (DADs) and transient inward current ( I ti) appeared in hypertrophied cells more often and were of larger amplitude than in control cells. DADs and I ti are carried principally by Na/Ca exchange with smaller contributions from a nonselective cation channel and from a Cl− channel. The latter is expressed only in hypertrophied myocytes. In hypertrophy, the density of caffeine-induced Na/Ca exchange current ( I Na/Ca) was increased by 26%, sarcoplasmic reticulum (SR) Ca2+ content as assessed from the integral of I Na/Ca was increased by 30%, the density of Na-pump current ( I pump) was reduced by 40%, and the intracellular Na+ content, measured by Na+-selective microelectrodes was increased by 55%. The results indicate that DADs and I ti are generated by spontaneous Ca2+ release from an overloaded SR caused by a downregulated Na pump and an upregulated Na/Ca exchange. These findings may explain the propensity for arrhythmias seen in this model of hypertrophy.

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.

Abstract 17193: Inhibiting Late Sodium Current Attenuates Isoproterenol-Induced Transient Inward Current and Delayed Afterdepolarizations in Ventricular Myocytes

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Yejia Song ◽  
Nesrine El-Bizri ◽  
Sridharan Rajamani ◽  
Luiz Belardinelli
Keyword(s):  
Ventricular Myocytes ◽  
Sodium Current ◽  
Selective Inhibition ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Cardiac Tissues ◽  
Inward Current ◽  
Transient Inward Current ◽  
Whole Cell Patch Clamp ◽  
Series Of Experiments

Introduction: The β-adrenergic agonist isoproterenol (ISO) is known to induce the arrhythmogenic transient inward current (I Ti ) and delayed afterdepolarization (DAD) via a stimulation of L-type Ca 2+ current. Recent studies found that ISO-induced DADs in cardiac tissues are inhibited by GS967, a selective blocker of the late Na + current (I NaL ). Thus, we hypothesize that I NaL contributes to the actions of ISO, and selective inhibition of this current will reduce ISO-induced I Ti and DADs. Methods: Transmembrane currents and action potentials of rabbit and guinea pig (GP) ventricular myocytes were recorded using the whole-cell patch-clamp technique. ISO (0.1 μM), GS967 (1 μM) and the Na + channel blocker tetrodotoxin (TTX, 3 μM) were used in the experiments. Results: In rabbit myocytes, application of ISO caused an increase in the amplitude of I NaL from -0.10±0.03 to -0.32±0.04 pA/pF (n = 17, p < 0.05). The ISO-stimulated I NaL was inhibited by GS967 and TTX. In one series of experiments, ISO increased the I NaL from -0.14±0.04 to -0.35±0.06 pA/pF, and GS967 applied in the presence of ISO reduced the current to -0.14±0.03 pA/pF (n = 9, p < 0.05). In another series of experiments, the amplitude of I NaL was increased by ISO from -0.17±0.08 to -0.41±0.09 pA/pF, and was decreased to -0.09±0.08 pA/pF when TTX was applied with ISO (n = 5, p < 0.05). Application of ISO also induced I Ti and DADs. GS967 applied in the presence of ISO inhibited the amplitude of I Ti by 52±6%, from -1.79±0.30 to -0.87±0.16 pA/pF (n = 8, p < 0.05). Consistent with the inhibition of I Ti , GS967 suppressed the amplitude of ISO-induced DADs by 56±12%, from 6.54±1.59 to 3.22±1.27 mV (n = 5, p < 0.05). Similarly, in GP myocytes ISO-induced I Ti and DADs were decreased by GS967 from -1.14±0.21 to -0.73±0.16 pA/pF (n = 7, p < 0.05) and from 7.16±0.59 to 4.67±0.24 mV (n = 5, p < 0.05), respectively. Conclusions: An increased I NaL is likely to contribute to the proarrhythmic effects of ISO in cardiac myocytes. GS967 significantly attenuated ISO-induced I NaL , I Ti and DADs, suggesting that inhibiting this current could be an effective strategy to antagonize the arrhythmogenic actions of β-adrenergic stimulation.

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.

Properties of voltage-gated Ca2+ channels in rabbit ventricular myocytes expressing Ca2+ channel α1E cDNA

2001 ◽  
Vol 280 (1) ◽  
pp. C175-C182 ◽  
Author(s):  
Michihiro Tateyama ◽  
Shuqin Zong ◽  
Tsutomu Tanabe ◽  
Rikuo Ochi
Keyword(s):  
Cardiac Myocytes ◽  
Fluorescent Protein ◽  
Ventricular Myocytes ◽  
Foreign Gene ◽  
Adrenergic Stimulation ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Whole Cell Patch Clamp ◽  
Green Fluorescent ◽  
Ca2 Channels

Using the whole-cell patch-clamp technique, we have studied the properties of α1ECa2+ channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current ( I Ca,L). Expression of green fluorescent protein significantly decreased the I Ca,L. By contrast, expression of α1E with β2b and α2/δ significantly increased the total Ca2+ current, and in these cells a Ca2+ antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni2+and was little affected by changing the charge carrier from Ca2+ to Ba2+ or by β-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of α1E did not generate T-like current in cardiac myocytes. On the other hand, expression of α1E decreased I Ca,L and slowed the I Ca,L inactivation. This inactivation slowing was attenuated by the β2b coexpression, suggesting that the α1E may slow the inactivation of I Ca,L by scrambling with α1C for intrinsic auxiliary β.

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.

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