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Intracellular magnesium during ischemia and reperfusion
J Mol Cell Cardiol21
(Supplement
II) (1989)
328
A BLOCK OF THE Na+/H+ EXCHANGE PREVENTS INITIATION OF THE REPERFUSION INDUCED FIBRILLATIONS. L.A. Vasilets, V.P. Mokh, B.I. Khodorov. Institute of Chemical Physics, Chemogolovka, Moscow region, Vishnevsky Surgery Institute, Moscow. A role of postischemic activation of Na+/H+ exchange in initiation of the rapid and severe reperfusion fibrillations was examined in experiments on the isolated, perfused by Langendorf rat hearts, using Na+/H+ exchange blocker ethylisopropylamiloride @PA). 5 pM of this compound effectively abolished postischemic fibrillations, observed after removal of 10 minutes ligation of the left anterior descending coronary artery, reducing the incidence of severe fibrillations from 60% in control to 8% with preparation and caused a 5-fold increase in time to their beginning. The same dose of ElPA blocked a recovery of contractile response and action potential (AP) duration shortening, showing a complete block of activation of Na+/H+ exchange, caused by washout of 20 mM NH&l, but did not change the AP parameters and contractile force at normal conditions. Thus, the activation of Na+/H+ exchange appears to be one of the principal mechanisms in the initiation of reperfusion induced arrhythmias.
329
FURAPTRA, A NEW FLUORESCENT INDICATOR FOR MEASURING CYTOSOLIC FREE MAGNESIUM (Mgi). Laboratory of E. Murphy, C.C. Freudenrich, L.A. Levy, M. Lieberman, R.E. London. Molecular Biophysics, NIEHS, POB 12233, Research Triangle Park, NC 27709 and Department of Cell Biology, Duke University Medical Center, Durham, NC 27710. Although Mgi is an important regulator of many cardiac cell functions, little is known about the concentration of Mgi and how it is regulated. We have recently developed a fluorescent indicator, FURAPTRA, to measure Mgiw FURAPTBA has s KMg of 1.5 mM and, upon binding Mg, it exhibits an excitation shift similar to the furaexcitation shift with Ca binding. Thus, Mgi can be measured using a ratio method, analogous to that for fura-2. FURAPTRA can be loaded into cells as the acetoxymethyl to physiologiester, and with a pK of 5.0 and a KCa of 53 uM FURAPTRA is insensitive cal variations in pH and Csi. Mgi was measured in FURAPTRA loaded cultured chick heart cells grown on coverslips by following emission fluorescence in response to simultaneous excitation at 335 and 370 nm. The basal Mgi, which averaged 0.48 2 0.03 (n=31) increased three-fold on Na-free perfusion. This increase could not simply be attributed to Nsi-Mgo exchange because a similar increase in Mgi occurred with Mgfree, Na-free perfusion. Futhermore, the increase in Mgi was largely attenuated when calcium was removed from the Na-free perfusate'. These data indicate an interaction (ML and CFF supported by HL07101, HL27105 and HL17670). between Cai sod Mgi,
330
INTRACELLULAR MAGNESIUM DURING ISCHEMIA AND REPERFUSION. J.H. Kirk&, C.J.A. van Echteld, T.J.C. Ruigrok, F.L. Meijler, Interuniversity Cardiology Institute and Department of Cardiology, University Hospital, Utrecht, The Netherlands. Loss of magnesium from ischemic myocardial tissue may impair the regulatory role of intracellular free magnesium (Mgf) in cellular biochemical and physiological processes. We used erP NMR spectroscopy in isolated rat hearts to study energy metabolism and pH and to calculate values for Mgf during control perfusion and ischemia, based on the chemical shift difference of the n- and g-phosphate of ATP 6x-d as a result of binding of ATP to Mg. During control perfusion ATP was found to be bound to Mg for 91%. After 15 min of total ischemia, when ATP was reduced to 2506, the fraction of MgATP slightly decreased to 88%. Assuming a Ko for MgATP of 0.038 pM and correcting the KD for the intracellular pH, these values corresponded with a Mgr of 0.36 (control) and 1.04 mM (15 min ischemia). This increase does not cover the much greater amount of Mg liberated from ATP at that time, suggesting either (temporary) binding to other intracellular sites or loss of Mg from the ischemic cells. However, during reperfusion after 30 min of ischemia we did not observe wash-out of magnesium. This indicates that in this model postischemic recovery is not limited by intracellular magnesium and that any beneficial effects of high Mg during reperfusion may be related to sarcolemmal rather than intracellular sites. s.110
(Supplement
II) (1989)
328
A BLOCK OF THE Na+/H+ EXCHANGE PREVENTS INITIATION OF THE REPERFUSION INDUCED FIBRILLATIONS. L.A. Vasilets, V.P. Mokh, B.I. Khodorov. Institute of Chemical Physics, Chemogolovka, Moscow region, Vishnevsky Surgery Institute, Moscow. A role of postischemic activation of Na+/H+ exchange in initiation of the rapid and severe reperfusion fibrillations was examined in experiments on the isolated, perfused by Langendorf rat hearts, using Na+/H+ exchange blocker ethylisopropylamiloride @PA). 5 pM of this compound effectively abolished postischemic fibrillations, observed after removal of 10 minutes ligation of the left anterior descending coronary artery, reducing the incidence of severe fibrillations from 60% in control to 8% with preparation and caused a 5-fold increase in time to their beginning. The same dose of ElPA blocked a recovery of contractile response and action potential (AP) duration shortening, showing a complete block of activation of Na+/H+ exchange, caused by washout of 20 mM NH&l, but did not change the AP parameters and contractile force at normal conditions. Thus, the activation of Na+/H+ exchange appears to be one of the principal mechanisms in the initiation of reperfusion induced arrhythmias.
329
FURAPTRA, A NEW FLUORESCENT INDICATOR FOR MEASURING CYTOSOLIC FREE MAGNESIUM (Mgi). Laboratory of E. Murphy, C.C. Freudenrich, L.A. Levy, M. Lieberman, R.E. London. Molecular Biophysics, NIEHS, POB 12233, Research Triangle Park, NC 27709 and Department of Cell Biology, Duke University Medical Center, Durham, NC 27710. Although Mgi is an important regulator of many cardiac cell functions, little is known about the concentration of Mgi and how it is regulated. We have recently developed a fluorescent indicator, FURAPTRA, to measure Mgiw FURAPTBA has s KMg of 1.5 mM and, upon binding Mg, it exhibits an excitation shift similar to the furaexcitation shift with Ca binding. Thus, Mgi can be measured using a ratio method, analogous to that for fura-2. FURAPTRA can be loaded into cells as the acetoxymethyl to physiologiester, and with a pK of 5.0 and a KCa of 53 uM FURAPTRA is insensitive cal variations in pH and Csi. Mgi was measured in FURAPTRA loaded cultured chick heart cells grown on coverslips by following emission fluorescence in response to simultaneous excitation at 335 and 370 nm. The basal Mgi, which averaged 0.48 2 0.03 (n=31) increased three-fold on Na-free perfusion. This increase could not simply be attributed to Nsi-Mgo exchange because a similar increase in Mgi occurred with Mgfree, Na-free perfusion. Futhermore, the increase in Mgi was largely attenuated when calcium was removed from the Na-free perfusate'. These data indicate an interaction (ML and CFF supported by HL07101, HL27105 and HL17670). between Cai sod Mgi,
330
INTRACELLULAR MAGNESIUM DURING ISCHEMIA AND REPERFUSION. J.H. Kirk&, C.J.A. van Echteld, T.J.C. Ruigrok, F.L. Meijler, Interuniversity Cardiology Institute and Department of Cardiology, University Hospital, Utrecht, The Netherlands. Loss of magnesium from ischemic myocardial tissue may impair the regulatory role of intracellular free magnesium (Mgf) in cellular biochemical and physiological processes. We used erP NMR spectroscopy in isolated rat hearts to study energy metabolism and pH and to calculate values for Mgf during control perfusion and ischemia, based on the chemical shift difference of the n- and g-phosphate of ATP 6x-d as a result of binding of ATP to Mg. During control perfusion ATP was found to be bound to Mg for 91%. After 15 min of total ischemia, when ATP was reduced to 2506, the fraction of MgATP slightly decreased to 88%. Assuming a Ko for MgATP of 0.038 pM and correcting the KD for the intracellular pH, these values corresponded with a Mgr of 0.36 (control) and 1.04 mM (15 min ischemia). This increase does not cover the much greater amount of Mg liberated from ATP at that time, suggesting either (temporary) binding to other intracellular sites or loss of Mg from the ischemic cells. However, during reperfusion after 30 min of ischemia we did not observe wash-out of magnesium. This indicates that in this model postischemic recovery is not limited by intracellular magnesium and that any beneficial effects of high Mg during reperfusion may be related to sarcolemmal rather than intracellular sites. s.110