A. Leyssens et al., THE RELATIONSHIP BETWEEN MITOCHONDRIAL STATE, ATP HYDROLYSIS, [MG2+],AND [CA2+](I) STUDIED IN ISOLATED RAT CARDIOMYOCYTES, Journal of physiology, 496(1), 1996, pp. 111-128
1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magne
sium concentration rises upon ATP hydrolysis. We have therefore used t
he Mg2+-sensitive fluorescent indicator Magnesium Green (MgG) to provi
de 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 rig
or, which signals ATP depletion. Contamination of the MgG signal by ch
anges in intracellular free Ca2+ concentration (the K-D of MgG for Ca2
+ is 4.7 mu M) was excluded by simultaneous measurement of [Ca2+](i) a
nd [Mg2+](i) cells dual loaded with fura-2 and MgG. The response to FC
CP was independent of external Mg2+, confirming an intracellular sourc
e for the rise in [Mg2+](i). 3. Simultaneous measurements of mitochond
rial NAD(P)H autofluorescence and mitochondrial potential (Delta psi(m
); JC-1 fluorescence) and of autofluorescence and MgG: allowed closer
study of the relationship between [Mg2+](i) and mitochondrial state. O
ligomycin abolished the FCCP-induced rise in [Mg2+]i without altering
the change in autofluorescence. Thus, the rise in [Mg2+](i) in respons
e to FCCP is consistent with the release of intracellular Mg2+ followi
ng ATP hydrolysis by the mitochondrial F1F0-ATPase, 4. The rise in [Mg
2+](i) was correlated with cell-attached recordings of ATP-sensitive K
+ channel (K-ATP) activity. In response to FCCP, an increase in K-ATP
channel activity was seen only as [Mg2+](i) reached a plateau. In resp
onse to blockade of mitochrondrial respiration and glycolysis with cya
nide (CN-) and 2-deoxyglucose (DOG), [Mg2+](i) rose more slowly but ag
ain K-ATP channel opening increased only when [Mg2+](i) readied a plat
eau and the cells shortened. 5. Oligomycin decreased the rate of rise
of [Mg2+](i), delayed the onset of rigor and increased the rate of mit
ochondrial depolarization in response to CN--DOG. Thus, with blockade
of mitochondrial respiration, Delta psi(m) is maintained by the mitoch
ondrial 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 recove
ry 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.