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Publication Detail
The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes.
  • Publication Type:
    Journal article
  • Publication Sub Type:
    Journal Article
  • Authors:
    Leyssens A, Nowicky AV, Patterson L, Crompton M, Duchen MR
  • Publication date:
    01/10/1996
  • Pagination:
    111, 128
  • Journal:
    J Physiol
  • Volume:
    496 ( Pt 1)
  • Status:
    Published
  • Country:
    England
  • Print ISSN:
    0022-3751
  • Language:
    eng
  • Keywords:
    Adenosine Triphosphate, Animals, Antimetabolites, Calcium, Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone, Cells, Cultured, Electrophysiology, Fluorescent Dyes, Heart, In Vitro Techniques, Magnesium, Male, Mitochondria, Heart, Myocardium, Oxidation-Reduction, Patch-Clamp Techniques, Potassium Channels, Proton-Translocating ATPases, Rats, Rats, Sprague-Dawley
Abstract
1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg(2+)-sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+]i rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the KD of MgG for Ca2+ is 4.7 microM) was excluded by simultaneous measurement of [Ca2+]i and [Mg2+]i in cells dual loaded with fura-2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+]i. 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (delta psi m; .-1 fluorescence) and of autofluorescence and MgG allowed closer study of the relationship between [Mg2+]i and mitochondrial state. Oligomycin abolished the FCCP-induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+]i in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0-ATPase. 4. The rise in [Mg2+]i was correlated with cell-attached recordings of ATP-sensitive K+ channel (KATP) activity. In response to FCCP, an increase in KATP channel activity was seen only as [Mg2+]i reached a plateau. In response to blockade of mitochondrial respiration and glycolysis with cyanide (CN-) and 2-deoxyglucose (DOG), [Mg2+]i rose more slowly but again KATP channel opening increased only when [Mg2+]i reached a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+]i delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN-_DOG. Thus, with blockade of mitochondrial respiration delta psi m is maintained by the mitochondrial F1F0-ATPase at the expense of ATP reserves. 6. In response to CN-_DOG, the initial rise in [Mg2+]i was accompanied by a small rise in [Ca2+]i. After [Mg2+]i reached a plateau and rigor developed, [Ca2+]i rose progressively. On reperfusion, in hypercontracted cells, [Ca2+]i recovered before [Mg2+]i and [ca2+]i oscillations were sustained while [Mg2+]i decreased. Thus on reperfusion, full recovery of [ATP]i is slow, but the activation of contractile elements and the restoration of [Ca2+]i does not require the re-establishment of millimolar concentrations of ATP.
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