Quantification of Ca2+ uptake in the sarcoplasmic reticulum of trout ventricular myocytes

Leif Hove-Madsen, Anna Llach, Lluis Tort

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    Abstract

    We measured Ca2+ uptake by the sarcoplasmic reticulum (SR) in trout ventricular myocytes, measuring indo 1 fluorescence in permeabilized cells or ionic currents in single myocytes subjected to voltage clamp. Titration of the SR Ca2+ pumps with thapsigargin gave a pump site density of 454 pmol/mg cell protein. Lowering the temperature from 20°C to 10 or 5°C reduced the SR Ca2+ uptake rate in permeabilized myocytes by 50 and 63%, respectively. Surprisingly, Ca2+ leak from the SR also decreased with decreasing temperatures. Exposure of single myocytes to 10 mM caffeine (Caf) induced a cell contracture and an inward ionic current. Neither contracture nor current decreased significantly after rest periods of 120 and 320 s. The inward current was due to Ca2+ extrusion by the Na+/Ca2+ exchanger (NCX), and the time integral of the exchange current (I(NCX)) was used to calculate the SR Ca2+ content. This gave a steady-state SR Ca2+ content of 22.5 ± 2.8 amol Ca2+/pF or 750 μM. When the SR was loaded by depolarizing the cell to +50 mV, the Ca2+ content increased with increasing length of the depolarization, reaching a maximum of 52.0 ± 5.9 amol Ca2+/pF. When the cell was depolarized to different voltages for 3 s, a subsequent Caf-induced I(NCX) increased with increasing voltage. At + 100 mV, the Ca2+ content was 36.6 ± 3.8 amol/pF, giving a maximal SR Ca2+ uptake rate of 12.2 ± 1.2 amol Ca2+ · pF-1·s-1 or 417 μM/s. We conclude that maximal SR Ca2+ content and Ca2+ uptake rates can be estimated using specific SR Ca2+ loading protocols. Contrary to the general assumption that contraction in lower vertebrates depends largely on transsarcolemmal Ca2+ fluxes, we found that although the L-type Ca2+ current is insufficient to fully activate contraction, the SR is capable of participating in the regulation of the cytosolic Ca2+ during the excitation-contraction coupling in trout ventricular myocytes.
    Original languageEnglish
    JournalAmerican Journal of Physiology - Regulatory Integrative and Comparative Physiology
    Volume275
    Issue number6 44-6
    Publication statusPublished - 1 Dec 1998

    Keywords

    • Caffeine
    • Calcium current
    • Calcium pump
    • Excitation-contraction coupling
    • Lower vertebrate heart
    • Sodium ion/calcium ion exchange

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  • Cite this

    Hove-Madsen, L., Llach, A., & Tort, L. (1998). Quantification of Ca2+ uptake in the sarcoplasmic reticulum of trout ventricular myocytes. American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 275(6 44-6).