Effects of substrate-free anoxia and veratridine on intracellular calcium concentration in isolated rat ventricular cardiomyocytes |
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Authors: | U M Rose P Couwenberg J W C M Jansen R J M Bindels C H Van Os |
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Institution: | (1) Department of Cell Physiology, University of Nijmegen, Trigon Bldg, P. O. Box 9101, NL-6500 HB Nijmegen, The Netherlands;(2) Department of Vascular Pharmacology, Solvay Duphar BV, P. O. Box 900, NL-1380 DA Weesp, The Netherlands |
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Abstract: | Cytosolic free Ca2+ concentration (Ca2+]i) was measured in freshly isolated rat ventricular cardiomyocytes during substrate-free anoxia. Cardiomyocytes were loaded with fura-2 and incubated in an anoxic chamber in which a pO2 equal to 0 mmHg was realized by inclusion of Oxyrase. Ca2+]i was measured in individual cells using digital imaging fluorescence microscopy. During anoxia, the shape of cardiomyocytes changed from a relaxed-elongated form into a rigor configuration within 15 min after the onset of anoxia. After the cells had developed the rigor state, a delayed rise in Ca2+]i reached a stable maximal level within 45 min. The mean values for the pre-anoxic and maximal anoxic Ca2+
i were 52±3 nM (N=42) and 2115±59 nM (N=45), respectively. The purported Na+ overload blocker R 56865, significantly reduced maximal anoxic Ca2+]i to 553±56 nM (P<0.05), implicating a role of elevated intracellular Na+ in the anoxia-induced increase in Ca2+]i. Veratridine (30 M), which induces Na+ overload, increased Ca2+]i to 787±39 nM. The compound R 56865 reduced veratridine-induced increases in Ca2+]i to 152±38 nM. Upon reperfusion, after 45 min of anoxia, two distinct responses were observed. Most often, Ca2+]i decreased upon reperfusion without a change in morphology or viability, while in the minority of cases, Ca2+]i increased further followed by hypercontraction and loss of cell viability. The mean value for Ca2+]i 10 min after reperfusion of the former group, was 752±46 nM (N=38). The cardiomyocyte cell shape could be followed by monitoring changes in the total fura-2 fluorescence (340+380 nm signal). Within 15 min after the onset of anoxia, the total fluorescence signal increased suddenly, before Ca2+]i started to rise, coinciding with the onset of rigor contraction induced by ATP depletion. |
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Keywords: | Ischaemia Ca2+ overload Cell injury Cell volume R 56865 |
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