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Relationship of inorganic phosphate and hypoxic failure in isolated perfused rat hearts
J Mol
Cell
Cardiol20
(Supplement
V) (1988)
10
MITOCHONDRIAL PHOSPHATE IS VISIBLE IN NMR SPECTRA OF RAT HEARTS. Pamela B Garlick, "Sibylle Soboll and '"Gillian R Bullock, Radiological Sciences, UMDS, Guy's Campus, London, UK, *Physiologische Chemie, Univ. Dusseldorf, FRG, "Ciba-Geigy, Basle, CH. Isolated rat hearts were subjected to aerobic perfusion at 37oC with PI-free Krebs for 40min (controls) followed by valinomycin (val) infusion (final cont. 13 nM) in the treated group; 31-P NMR spectra were acquired at 162MHz (300,700,0.38s pulses). In control spectra, 2 peaks were observed in the P.I region at 2.38 + 0.01 and 2.79 ~0.01 ppm (n=18, + SEM, PCr at -2.52ppm) and it was thought that the 2.8ppm peak might arise from the mltochondrial (mito) P,. Upon val infusion, the 2.8ppm peak increased such that by 12 min it was the same size as the 2.4ppm peak. Control and val hearts after 12 min (n=18 in total) were divided into 3 groups and: 1. freeze-clamped and extracted with perchloric acid (PCA) to disrupt the sub-cellular organelles; 2. freeze-clamped, ground, lyophilized and fractionated in non-aqueous solvents: 3. perfusion-fixed for electron microscopy (EM). In all PCA extract spectra, there was only ONE Pi peak (2.4ppm, buffer pH 7.0). From the fractionation studies (assayed blind), there were 1.8 (range 1.5-2.1) and 3.5 (range 3.0-3.8) micromoles mito P, per g in control and val hearts, respectively. The EM studies (assayed blind) showed that mito swelling, with a mean volume increase of 31%, occurred in 50% of the cells in val hearts. In conclusion, having quantified the increased mito Pi and volume in valinomycin-treated hearts, we assign the peak at 2.8ppm in 31-P NMR spectra to the mitochondrial P,.
11
RELATIONSHIP OF INORGANIC PHOSPHATE AND HYPOXIC FAILURE IN ISOLATED PERFUSED RAT HEARTS. Ii. Kammermeier, E. Roeb, V. Perlitz, T. Wiedemann. Department of Physiology, Medical Faculty, Technical University, Aachen, F.R.G. The relationship of inorganic cytoplasmic phosphate (Pi) and isovolumic peak systolic pressure (Psrst) was investigated in 4 experimental approaches in hypoxic rat hearts. Methods: Isolated rat hearts with intraventricular balloon perfused with Krebs-Ringer-medium (11 mM glucose) gassed with 95% to 8% 02 (5% COz, complementary Ns). Measurement of Psrat, and chemical assay, freeze stop, HPLC, 3 rain after comencement of hypoxia. Pi calculated respecting published data on compartmentation. 1. With graded hypoxia there was a negative apparent relationship between Pi (4 - 12 mM) and Psvst (120 - 20 mmlig). 2. With graded reoxygenation a similar relationship was observed but Pi values were significantly higher by about 5 mM at similar Ps~s~ values. 3. During inotropic interventions in hypoxia there was a positive relationship of Pi v. Psvst. 4. During experimental change of heart rate in hypoxia (80 - 400/min) Psvst varied over a wide range (30 - 140 mmHg) with high Pi (12 - 14 mM). In normoxic controls (150 - 4OO/min) P sYSt varied (95 - 135 mmHg) with low Pi 4.5 mM. It is concluded that cytoplasmic Pi is not the only and not the primary determinant factor for hypoxic failure. (Supported by the DFG).
12
FREE ENERGY OF ATP-HYDROLYSIS (dG/dE;) AND CARDIAC PERFORMANCE DURING GRADED HYPOXIA, GRADED REOXYGENATION AND INOTROPIC INTERVENTIONS. Ii. Kammermeier, E. Roeb, Th. Wiedemann. Department of Physiology, Medical Faculty, Technical University, Aachen, F.R.G. The relationship between dG/dg and peak systolic pressure (Psyst) was investigated in isolated isovolumically working (intraventricular balloon) perfused rat hearts. Hypoxia: medium gased with 95 to 8% 0s (5% COz, complementary Nz)Biochemicalculated from cytosolic [ADP] using CK cal assay after freeze stop by HPLC. dG/dl equilibrium equation. - During graded hypoxia there was an apparent positive relationship between Psvst (140 to 35 mm Hg) and dG/de (-60 to -45 kJ/mol) - during graded reoxygenation a similar relationship was found as in graded hypoxia - with experimentally changed heart rate (400 to 8O/minf during hypoxia a pronounced negative force frequency relationship was accompanied by largely constant dG/dg values at the respective levels of hypoxia. - A tendency towards a negative relationship between dG/dg and Psvst was observed during positive inotropic interventions in hypoxia. The observations can be interpreted in terms of free energy dependence of processes This dependence could act as a protective mechaof excitation contraction coupling. nism which prevents lowering of free energy level critical for survival of the cell at costs of mechanic performance. (Supported by DFG). s.22
Cell
Cardiol20
(Supplement
V) (1988)
10
MITOCHONDRIAL PHOSPHATE IS VISIBLE IN NMR SPECTRA OF RAT HEARTS. Pamela B Garlick, "Sibylle Soboll and '"Gillian R Bullock, Radiological Sciences, UMDS, Guy's Campus, London, UK, *Physiologische Chemie, Univ. Dusseldorf, FRG, "Ciba-Geigy, Basle, CH. Isolated rat hearts were subjected to aerobic perfusion at 37oC with PI-free Krebs for 40min (controls) followed by valinomycin (val) infusion (final cont. 13 nM) in the treated group; 31-P NMR spectra were acquired at 162MHz (300,700,0.38s pulses). In control spectra, 2 peaks were observed in the P.I region at 2.38 + 0.01 and 2.79 ~0.01 ppm (n=18, + SEM, PCr at -2.52ppm) and it was thought that the 2.8ppm peak might arise from the mltochondrial (mito) P,. Upon val infusion, the 2.8ppm peak increased such that by 12 min it was the same size as the 2.4ppm peak. Control and val hearts after 12 min (n=18 in total) were divided into 3 groups and: 1. freeze-clamped and extracted with perchloric acid (PCA) to disrupt the sub-cellular organelles; 2. freeze-clamped, ground, lyophilized and fractionated in non-aqueous solvents: 3. perfusion-fixed for electron microscopy (EM). In all PCA extract spectra, there was only ONE Pi peak (2.4ppm, buffer pH 7.0). From the fractionation studies (assayed blind), there were 1.8 (range 1.5-2.1) and 3.5 (range 3.0-3.8) micromoles mito P, per g in control and val hearts, respectively. The EM studies (assayed blind) showed that mito swelling, with a mean volume increase of 31%, occurred in 50% of the cells in val hearts. In conclusion, having quantified the increased mito Pi and volume in valinomycin-treated hearts, we assign the peak at 2.8ppm in 31-P NMR spectra to the mitochondrial P,.
11
RELATIONSHIP OF INORGANIC PHOSPHATE AND HYPOXIC FAILURE IN ISOLATED PERFUSED RAT HEARTS. Ii. Kammermeier, E. Roeb, V. Perlitz, T. Wiedemann. Department of Physiology, Medical Faculty, Technical University, Aachen, F.R.G. The relationship of inorganic cytoplasmic phosphate (Pi) and isovolumic peak systolic pressure (Psrst) was investigated in 4 experimental approaches in hypoxic rat hearts. Methods: Isolated rat hearts with intraventricular balloon perfused with Krebs-Ringer-medium (11 mM glucose) gassed with 95% to 8% 02 (5% COz, complementary Ns). Measurement of Psrat, and chemical assay, freeze stop, HPLC, 3 rain after comencement of hypoxia. Pi calculated respecting published data on compartmentation. 1. With graded hypoxia there was a negative apparent relationship between Pi (4 - 12 mM) and Psvst (120 - 20 mmlig). 2. With graded reoxygenation a similar relationship was observed but Pi values were significantly higher by about 5 mM at similar Ps~s~ values. 3. During inotropic interventions in hypoxia there was a positive relationship of Pi v. Psvst. 4. During experimental change of heart rate in hypoxia (80 - 400/min) Psvst varied over a wide range (30 - 140 mmHg) with high Pi (12 - 14 mM). In normoxic controls (150 - 4OO/min) P sYSt varied (95 - 135 mmHg) with low Pi 4.5 mM. It is concluded that cytoplasmic Pi is not the only and not the primary determinant factor for hypoxic failure. (Supported by the DFG).
12
FREE ENERGY OF ATP-HYDROLYSIS (dG/dE;) AND CARDIAC PERFORMANCE DURING GRADED HYPOXIA, GRADED REOXYGENATION AND INOTROPIC INTERVENTIONS. Ii. Kammermeier, E. Roeb, Th. Wiedemann. Department of Physiology, Medical Faculty, Technical University, Aachen, F.R.G. The relationship between dG/dg and peak systolic pressure (Psyst) was investigated in isolated isovolumically working (intraventricular balloon) perfused rat hearts. Hypoxia: medium gased with 95 to 8% 0s (5% COz, complementary Nz)Biochemicalculated from cytosolic [ADP] using CK cal assay after freeze stop by HPLC. dG/dl equilibrium equation. - During graded hypoxia there was an apparent positive relationship between Psvst (140 to 35 mm Hg) and dG/de (-60 to -45 kJ/mol) - during graded reoxygenation a similar relationship was found as in graded hypoxia - with experimentally changed heart rate (400 to 8O/minf during hypoxia a pronounced negative force frequency relationship was accompanied by largely constant dG/dg values at the respective levels of hypoxia. - A tendency towards a negative relationship between dG/dg and Psvst was observed during positive inotropic interventions in hypoxia. The observations can be interpreted in terms of free energy dependence of processes This dependence could act as a protective mechaof excitation contraction coupling. nism which prevents lowering of free energy level critical for survival of the cell at costs of mechanic performance. (Supported by DFG). s.22