Calcium currents of isolated bovine ventricular myocytes are fast and of large amplitude View Full Text


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Article Info

DATE

1982-10

AUTHORS

Gerrit Isenberg, Udo Klöckner

ABSTRACT

A two microelectrode voltage clamp technique was applied to ventricular myocytes superfused with Tyrode solution containing 1.8 or 3.6 mM [Ca]0. The clamp settled a 100 mV step up over a “capacitive membrane area” of 2×10−4 cm2 within 200 μs; the capacitive current peaked within 60 μs and decayed afterwards with a τc of 60 μs, indicating a non-distributed series resistance of less than 30 Ohm·cm2.Clamping from resting potential (−80 mV) to 0 mV evoked the 2 inward current componentsINa andICa.INa was greater than 50 nA and prevented adequate voltage control during the initial 2 ms; it could be blocked by 60 μM TTX, sodium removal or by clamping from a conditioning pre-step of −50 mV.ICa remained essentially unaltered by the 3 procedures listed above, but could be blocked by 2 μM D600, by 5 mM Ni or by 5 mM Co.Clamping from −50 to 0 mV evoked a net inward current which peaked within 2 ms to −8 nA and changed 200 ms later into a net outward current. Plotting the time dependent values on semilog paper, 3 time constants became apparent. From tail current analysis and sensitivity to D600 we attribute the slow exponential (τ≈1s) to activation of potassium current (Ix), and the 2 faster exponentials (τ ≈ 1 s) to inactivation ofICa.ICa was defined by the time course of inactivation. At 0 mV, it had a peak amplitude of 34±12 μA/cm2 in 1.8 mM [Ca]0. Doubling [Ca]0 to 3.6 mM increased peakICa to 42±10 μA/cm2. Addition of 0.2 μM adrenaline increased peakICa up to 80 μA/cm2. The estimates of peakICa were about 20% greater whenICa was defined by its sensitivity to D 600.Voltage dependence.ICa had a threshold at about −35 mV, reached a maximum around +5 mV and declined again for more positive potentials. Between +50 and +60 mV the peak changed from a (net) inward to a net outward current; this could indicate an “apparent” reversal potential (Erev) but also masking ofICa by a transient outward current.Activation. By dividing peakICa by (V-Erev) the steady-state activation curve was estimated. The data could be fitted according to\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\bar g/(1 + exp(V - V_h )/k)$$ \end{document} using\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\bar g = 0.76 \pm 0.25 mS/cm^2$$ \end{document},k=9±1.5 mV andVh=−18±4 mV. Activation time course could be fitted with a single exponential, τd being 1.1 ms close to threshold and 0.5 ms at +10 mV. Deactivation occurred with a similar fast time course.Inactivation. Steady-state inactivation was evaluated by stepping from more and more positive holding potentials to +10 mV. The data were fitted according to\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\bar g/(1 + exp(V - V_h )/k)$$ \end{document} using\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\bar g = 0.98 \pm 0.11 mS/cm^2$$ \end{document},k=−9±1.1 mV andVh=−22±3 mV. The inactivation time course was described with 2 exponentials\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\tau _{f_2 }$$ \end{document} and\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\tau _{f_2 } \cdot \tau _{f_1 }$$ \end{document} was about 5 ms around threshold, for more positive potentials it increased monotonic.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\tau _{f_2 }$$ \end{document} was 80 ms near threshold, 30–40 ms around +10 mV and increased towards 100 ms for +40 mV giving a U-shaped voltage dependence.In Comparison with the slow inward current (isi) reported in the literature,ICa activates faster and has a larger amplitude. We explain the apparent inconsistency by differences in the series resistanceRs. AddingRs of 1.7 kOhm·cm2 between ground and bath, reduced peakICa and prolonged the “activation” time course. In case of previous experiments where isolated myocytes were investigated with a single microelectrode voltage clamp (Isenberg and Klöckner 1980),Rs resulted from the incompletely compensated electrode resistance. In case of multicellular heart preparationsRs may result from endothelium and the extracellular cleft space. More... »

PAGES

30-41

References to SciGraph publications

  • 1977-01. Cardiac Purkinje fibres in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1980-03. Glycocalyx is not required for slow inward calcium current in isolated rat heart myocytes in NATURE
  • 1982-10. Calcium tolerant ventricular myocytes prepared by preincubation in a “KB medium” in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1982-10. Isolated bovine ventricular myocytes in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1975-03. Kinetics of inactivation and recovery of the slow inward current in the mammalian ventricular myocardium in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1980-10. Slow inward current and its role mediating the chronotropic effect of epinephrine in the rabbit sinoatrfal node in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1975-03. Calcium conductance and tension in mammalian ventricular muscle in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1974-09. Iontophoretic application of acetylcholine: Advantages of high resistance micropipettes in connection with an electronic current pump in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1972-03. Inward membrane currents in mammalian myocardium in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1972-03. The ionic nature of slow inward current and its relation to contraction in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
  • 1979-05. Risk and advantages of using strongly beveled microelectrodes for electrophysiological studies in cardiac Purkinje fibers in PFLÜGERS ARCHIV - EUROPEAN JOURNAL OF PHYSIOLOGY
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    http://scigraph.springernature.com/pub.10.1007/bf00584965

    DOI

    http://dx.doi.org/10.1007/bf00584965

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/6294586


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        "description": "A two microelectrode voltage clamp technique was applied to ventricular myocytes superfused with Tyrode solution containing 1.8 or 3.6 mM [Ca]0. 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    25 schema:description A two microelectrode voltage clamp technique was applied to ventricular myocytes superfused with Tyrode solution containing 1.8 or 3.6 mM [Ca]0. The clamp settled a 100 mV step up over a “capacitive membrane area” of 2×10−4 cm2 within 200 μs; the capacitive current peaked within 60 μs and decayed afterwards with a τc of 60 μs, indicating a non-distributed series resistance of less than 30 Ohm·cm2.Clamping from resting potential (−80 mV) to 0 mV evoked the 2 inward current componentsINa andICa.INa was greater than 50 nA and prevented adequate voltage control during the initial 2 ms; it could be blocked by 60 μM TTX, sodium removal or by clamping from a conditioning pre-step of −50 mV.ICa remained essentially unaltered by the 3 procedures listed above, but could be blocked by 2 μM D600, by 5 mM Ni or by 5 mM Co.Clamping from −50 to 0 mV evoked a net inward current which peaked within 2 ms to −8 nA and changed 200 ms later into a net outward current. Plotting the time dependent values on semilog paper, 3 time constants became apparent. From tail current analysis and sensitivity to D600 we attribute the slow exponential (τ≈1s) to activation of potassium current (Ix), and the 2 faster exponentials (τ ≈ 1 s) to inactivation ofICa.ICa was defined by the time course of inactivation. At 0 mV, it had a peak amplitude of 34±12 μA/cm2 in 1.8 mM [Ca]0. Doubling [Ca]0 to 3.6 mM increased peakICa to 42±10 μA/cm2. Addition of 0.2 μM adrenaline increased peakICa up to 80 μA/cm2. The estimates of peakICa were about 20% greater whenICa was defined by its sensitivity to D 600.Voltage dependence.ICa had a threshold at about −35 mV, reached a maximum around +5 mV and declined again for more positive potentials. Between +50 and +60 mV the peak changed from a (net) inward to a net outward current; this could indicate an “apparent” reversal potential (Erev) but also masking ofICa by a transient outward current.Activation. By dividing peakICa by (V-Erev) the steady-state activation curve was estimated. The data could be fitted according to\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\bar g/(1 + exp(V - V_h )/k)$$ \end{document} using\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\bar g = 0.76 \pm 0.25 mS/cm^2$$ \end{document},k=9±1.5 mV andVh=−18±4 mV. Activation time course could be fitted with a single exponential, τd being 1.1 ms close to threshold and 0.5 ms at +10 mV. Deactivation occurred with a similar fast time course.Inactivation. Steady-state inactivation was evaluated by stepping from more and more positive holding potentials to +10 mV. The data were fitted according to\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\bar g/(1 + exp(V - V_h )/k)$$ \end{document} using\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\bar g = 0.98 \pm 0.11 mS/cm^2$$ \end{document},k=−9±1.1 mV andVh=−22±3 mV. The inactivation time course was described with 2 exponentials\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\tau _{f_2 }$$ \end{document} and\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\tau _{f_2 } \cdot \tau _{f_1 }$$ \end{document} was about 5 ms around threshold, for more positive potentials it increased monotonic.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$\tau _{f_2 }$$ \end{document} was 80 ms near threshold, 30–40 ms around +10 mV and increased towards 100 ms for +40 mV giving a U-shaped voltage dependence.In Comparison with the slow inward current (isi) reported in the literature,ICa activates faster and has a larger amplitude. We explain the apparent inconsistency by differences in the series resistanceRs. AddingRs of 1.7 kOhm·cm2 between ground and bath, reduced peakICa and prolonged the “activation” time course. In case of previous experiments where isolated myocytes were investigated with a single microelectrode voltage clamp (Isenberg and Klöckner 1980),Rs resulted from the incompletely compensated electrode resistance. In case of multicellular heart preparationsRs may result from endothelium and the extracellular cleft space.
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