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Adenine nucleotide metabolism in ischemic and reperfused rat hearts
J Mol Cell Cardiol21
(Supplement
II) (1989)
451 ADENINJZ NUCLEOTIDE
METABOLISM IN ISCHBMIC AND RBPERFUSED RAT HEARTS. M. van Bilsen, G.J. van der Vusse, W.A. Coumans, R.S. Reneman. Dept. of Physiology, University of Limburg, Maastricht, The Netherlands. Cessation of flow in isolated rat hearts caused a gradual decline in tissue ATP levels, a transient increase in ADP, and a slight but significant increase in AMP. At the same time adenine nucleotide degradation products, such as adenosine, inosine (major break-down product), hypoxanthine and xanthine accumulated in the ischemic tissue. When the time interval of ischemia exceeded 45 min AMP continued to rise, whereas the content of adenosine, inosine and (hypo)xanthine hardly changed. This finding points to constraints on AMP degradation and flux through the degradation pathway from adenosine to uric acid in the ischemic heart. In myocardial preparations, the cells of which were deliberately disrupted by freezing and thawing, AMP did not accumulate and adenine nucleotides were degraded down to hypoxanthine during anoxic incubation. These results strongly suggest that compartmentalization of substrates and enzymes is responsible for the observed preferential accumulation of AMP and inosine in “intact” ischemic tissue. Inhibition of hypoxanthine degradation is explained by the absence of oxygen, an essential cofactor for xanthine oxidase. After restoration of flow accumulated purines were released into the coronary effluent and, concomitantly, further metabolized. Comparison of tissue levels of hypoxanthine, xanthine and uric acid prior to reperfusion and the amounts released during reperfusion indicates that substantial amounts of potentially hazardous xanthine-oxidase derived reactive oxygen species are formed during the early reperfusion phase. (Supported by MEDIGON/NWO grant nr 900-516-091).
452 DIFFERENTIAL EFFECTS OF REOXYGENATION/RBPERFUSIONON INTRACELLULAR Ca2+ AND LEFT VENTRICULAR PRESSUREIN ISOLATED PERFUSEDFERRET HEARTS. Y. Kihara, W. Grossman, J. P. Morgan. Harvard-Thorndike Laboratory of Beth Israel Hospital, Boston. MA 02215 The purpose of this study was to determine whether changes in intracellular Ca2+
([Cz?],) can account for the contractile abnormalities that develop during reoxygenation (Re-ox)/reperfusion (Re-per) after global hypoxia (H)/ischemia (I). Hearts were isolated from male ferrets (n-10) and coronary perfused via the aorta with a bicarbonate buffered salt solution bubbled with 95% 0,/5% CO, to pH 7.4 (T=30° C; Ca"=lmM) while pacing at 120 beats/min. We used the bioluminescent Ca2+ indicator aequorin to record [Ca'+], simultaneously with LVP (J Gen Physiol 1988; 92:47). H was induced by bubbling the perfusate with 95% N,/5% CO,; global I by clamping aortic inflow. Re-ox was accomplished by switching to 95% 0,/5% CO,; Re-per by releasing the aortic clamp. H decreased peak [Caz+li and LVP; these changes were reversed by Re-ox. In contrast, I decreased LVP but markedly increased [Ca'+],, changes that were reversed by Re-per after brief periods. However, the time courses of the [Ca'+], transients and LVP traces moved in opposite directions; they were respectively prolonged/abbreviated by both H and I and abbreviated/prolongedbyboth Re-ox/Re-per. These results indicate that the observed changes in LVP cannot be attributed alone to changes in [Caz+li but are caused, in part, by significant changes in myofilament Ca2+ sensitivity as well as by other factors determining cross-bridge interaction. (Support: HL31117; HL01611 to JPM and an AHA Fellowshin to YK ‘I
453
RATE OF DEVELOPMENT OF IRREVERSIBLE INJURY IN SEVERELY ISCHAEMIC CARDIAC CONDUCTION TISSUE. L.C. Armiger, D.A.I. Mathias. Department of Pathology, University of
Auckland School of Medicine,
Auckland,
New Zealand.
The fine structural alteration induced by severe ischaemia, and its potential reversibility, were studied in the atrioventricular junctional conduction tissues (AVJ) of the isolated rat heart. Langendorff preparations were made globally ischaemic for 0.5, 1, 2, 3 or 4 h, both with and without 30 min of subsequent reperfusion, and the AVJ examined by light and electron microscopy using a largespecimen resin-embedding method. After only 30 min of ischaemia nodal and bundle cells already showed marked intracellular oedema with loss of glycogen and swelling of organelles. These changes were somewhat more severe after l-3 h, but strongly aggregated nuclear chromatin and intramitochondrial dense inclusions were not prominent until after 4 h. Reperfusion after 0.5 - 2 h of ischaemia completely reversed the alterations in all or most cells, and after 3 h it reversed swelling in up to 50X of cells although these remained glycogen depleted. After 4 h, cellular damage persisted throughout reperfused tissue and large sarcolemmal blebs, together with clumping of myofibrillar material, were widespread. Severely ischaemic conducting cells therefore die at different rates but none survive longer than 4 h. s.151
(Supplement
II) (1989)
451 ADENINJZ NUCLEOTIDE
METABOLISM IN ISCHBMIC AND RBPERFUSED RAT HEARTS. M. van Bilsen, G.J. van der Vusse, W.A. Coumans, R.S. Reneman. Dept. of Physiology, University of Limburg, Maastricht, The Netherlands. Cessation of flow in isolated rat hearts caused a gradual decline in tissue ATP levels, a transient increase in ADP, and a slight but significant increase in AMP. At the same time adenine nucleotide degradation products, such as adenosine, inosine (major break-down product), hypoxanthine and xanthine accumulated in the ischemic tissue. When the time interval of ischemia exceeded 45 min AMP continued to rise, whereas the content of adenosine, inosine and (hypo)xanthine hardly changed. This finding points to constraints on AMP degradation and flux through the degradation pathway from adenosine to uric acid in the ischemic heart. In myocardial preparations, the cells of which were deliberately disrupted by freezing and thawing, AMP did not accumulate and adenine nucleotides were degraded down to hypoxanthine during anoxic incubation. These results strongly suggest that compartmentalization of substrates and enzymes is responsible for the observed preferential accumulation of AMP and inosine in “intact” ischemic tissue. Inhibition of hypoxanthine degradation is explained by the absence of oxygen, an essential cofactor for xanthine oxidase. After restoration of flow accumulated purines were released into the coronary effluent and, concomitantly, further metabolized. Comparison of tissue levels of hypoxanthine, xanthine and uric acid prior to reperfusion and the amounts released during reperfusion indicates that substantial amounts of potentially hazardous xanthine-oxidase derived reactive oxygen species are formed during the early reperfusion phase. (Supported by MEDIGON/NWO grant nr 900-516-091).
452 DIFFERENTIAL EFFECTS OF REOXYGENATION/RBPERFUSIONON INTRACELLULAR Ca2+ AND LEFT VENTRICULAR PRESSUREIN ISOLATED PERFUSEDFERRET HEARTS. Y. Kihara, W. Grossman, J. P. Morgan. Harvard-Thorndike Laboratory of Beth Israel Hospital, Boston. MA 02215 The purpose of this study was to determine whether changes in intracellular Ca2+
([Cz?],) can account for the contractile abnormalities that develop during reoxygenation (Re-ox)/reperfusion (Re-per) after global hypoxia (H)/ischemia (I). Hearts were isolated from male ferrets (n-10) and coronary perfused via the aorta with a bicarbonate buffered salt solution bubbled with 95% 0,/5% CO, to pH 7.4 (T=30° C; Ca"=lmM) while pacing at 120 beats/min. We used the bioluminescent Ca2+ indicator aequorin to record [Ca'+], simultaneously with LVP (J Gen Physiol 1988; 92:47). H was induced by bubbling the perfusate with 95% N,/5% CO,; global I by clamping aortic inflow. Re-ox was accomplished by switching to 95% 0,/5% CO,; Re-per by releasing the aortic clamp. H decreased peak [Caz+li and LVP; these changes were reversed by Re-ox. In contrast, I decreased LVP but markedly increased [Ca'+],, changes that were reversed by Re-per after brief periods. However, the time courses of the [Ca'+], transients and LVP traces moved in opposite directions; they were respectively prolonged/abbreviated by both H and I and abbreviated/prolongedbyboth Re-ox/Re-per. These results indicate that the observed changes in LVP cannot be attributed alone to changes in [Caz+li but are caused, in part, by significant changes in myofilament Ca2+ sensitivity as well as by other factors determining cross-bridge interaction. (Support: HL31117; HL01611 to JPM and an AHA Fellowshin to YK ‘I
453
RATE OF DEVELOPMENT OF IRREVERSIBLE INJURY IN SEVERELY ISCHAEMIC CARDIAC CONDUCTION TISSUE. L.C. Armiger, D.A.I. Mathias. Department of Pathology, University of
Auckland School of Medicine,
Auckland,
New Zealand.
The fine structural alteration induced by severe ischaemia, and its potential reversibility, were studied in the atrioventricular junctional conduction tissues (AVJ) of the isolated rat heart. Langendorff preparations were made globally ischaemic for 0.5, 1, 2, 3 or 4 h, both with and without 30 min of subsequent reperfusion, and the AVJ examined by light and electron microscopy using a largespecimen resin-embedding method. After only 30 min of ischaemia nodal and bundle cells already showed marked intracellular oedema with loss of glycogen and swelling of organelles. These changes were somewhat more severe after l-3 h, but strongly aggregated nuclear chromatin and intramitochondrial dense inclusions were not prominent until after 4 h. Reperfusion after 0.5 - 2 h of ischaemia completely reversed the alterations in all or most cells, and after 3 h it reversed swelling in up to 50X of cells although these remained glycogen depleted. After 4 h, cellular damage persisted throughout reperfused tissue and large sarcolemmal blebs, together with clumping of myofibrillar material, were widespread. Severely ischaemic conducting cells therefore die at different rates but none survive longer than 4 h. s.151