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Mechanism of cell damage in brain ischemia: A hypothesis
Life Sciences, Vol. 37, pp. 71-73 Printed in the U.S.A.
Pergamon Press
MECHANISM OF CELL DAMAGEIN BRAIN ISCHEMIA: A HYPOTHESIS K. Kariman, M.D. Box 3848, Duke University Medical Center, Durham, NC 27710 (Received in final form April 24, 1985)
Summary Mitochondria are known to develop a series of abnormalities as a result of ischemia. The i n a b i l i t y of mitochondria to resume normal function following reperfusion has been implicated as an important factor in irreversible cell damage. However, the mechanism of mitochondrial injury after ischemic brain insult is poorly understood. In this paper a hypothesis is proposed which concentrates on the interrelated roles of phosphate, calcium, and electron transport on ischemic brain cell injury. Ischemia is known to set rapidly into motion a complex series of events that involve v i r t u a l l y every organelle and subcellular system (1,2). Oxidative phosphorylation ceases, ATP stores are depleted, and v i r t u a l l y all energydependent functions cease. Although the mechanism(s) responsible for cell death in ischemic brain is poorly understood, i t is generally accepted that ischemic cell death is a consequence of mitochondrial injury (3). As recently reviewed by Siesjo (4,5) various factors (e.g. lactic acidosis, production of oxygen radicals, increased free fatty acids concentrations, etc.) have been hypothesized to be responsible for cell damage in brain ischemia. I t seems, however, that these factors are most l i k e l y the result, rather than the causes, of ischemic brain injury. In this communication a new hypothesis is proposed which focuses on the role of the triad of phosphate, calcium, and electron transport on ischemic cell injury and death in the brain. a) Role of phosphate: Dramatic increases in intracellular inorganic phosphate (Pi) in brain ischemia have been recently detected by nuclear magnetic resonance (NMR) technique in various laboratory animals (6-8). Pi, a potent agent, is known to induce mitochondrial swelling (9,10). Mitochondrial swelling is generally used as an index of mitochondrial dysfunction. I t has been demonstrated that respiration-dependent accumulation of potassium activated by Pi is responsible for the osmotic swelling of mitochondria (10). Studies carried out in the presence of external calcium indicate that Pi induces an energydissipating cycling of calcium across the inner membrane which is related to impaired oxidative phosphorylation (11). b) Role of calcium: At least in myocardial ischemia followed by reperfusion i t has been demonstrated that the resulting mitochondrial dysfunction appears to be mediated in part by calcium (12). Recently, decreased mitochondrial injury in response to inhibition of calcium transport has been demonstrated by Schwartz and associates (13). Using diltiazem, a calcium antagonist, these investigators were able to completely reverse the depression of state 3 mitochondrial respiratory rate observed following I0 minutes of coronary artery occlusion in dogs. Perhaps the most significant observation reported by this group (14) was the existence of two separable components of the Pi-induced 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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Brain Ischemia: Mechanism of Cell Damage
Vol. 37, No. i, 1985
mitochondriaI swelling. One was calcium antagonist ruthenium red (RR)insensitive which appears not to cause damage to mitochondria. The second component was RR-sensitive and results in inhibition and partial uncoupling of oxidative phosphorylation even in the absence of free extramitochondrial calcium. This strongly suggests the involvement of calcium in the Pi-induced damage to oxidative phosphorylation which may occur during ischemia. Furthermore, mitochondrial accumulation of calcium which is known to occur during reperfusion will further increase mitochondrial dysfunction and cellular injury (12). c) Role of electron transport: Based on observations made some 25 years ago in isolated rat liver mitochondria by Hunter et al (15), i t was clearly shown that electron transport was involved in phosphate-induced mitochondrial swelling. As proposed by these investigators, i t seems that electron transport is important for creating in the mitochondrial membrane a situation in which agents that cause swelling are able to produce their effect. Hunter et al (15) demonstrated that phosphate-induced swelling was prevented by inhibitors of electron transfer between pyridine nucleotides and oxygen as well as by the uncoupler 2,4-dinitrophenol. Using inhibitors of electron transport and 2,4-dinitrophenol in a model of perfused rat head we have observed improved mitochondrial redox responses to transient ischemic insults in situ (submitted for publication). An important observation was recently reported by Lange et al (16) in rabbit heart mitochondria. A rapid inactivation of oxidative phosphorylation was demonstrated when phosphate and oxygen were present. This inactivation was partially, but not completely, precluded by calcium antagonists. However, inactivation was prevented by incubation of mitochondria in the absence of oxygen, indicating that injury elicited by phosphate is oxygen dependent. Since intracellular Pi markedly increased with ischemia, reperfusion with oxygenated medium can paradoxically augment mitochondrial injury in this setting (17). The role of oxygen in phosphate-induced injury may be to provide an electron sink thus allowing transport into the mitochondrial matrix of phosphate and/or calcium, which could therefore be the primary mediators of injury (16). Through the same mechanism of inhibition of electron transport, anaerobiosis has been shown (9,15) to protect against v i r t u a l l y every agent known to induce mitochondrial swelling. Comments: I t is hypothesized that the triad of Pi, calcium, and electron transport is necessary for ischemic injury of the brain. The rise in intracellular Pi during ischemia, in the presence of an active electron transport, is the primary event responsible for mitochondrial edema and therefore dysfunction. Pi-induced mitochondrial injury, however, is in part mediated by calcium and calcium antagonists may have a role in preventing this phenomenon. A marked exaggeration of these phenomena occurs during reperfusion. In particular, increased mitochondrial accumulation of calcium during reperfusion results in additional cell injury. This hypothesis will explain the poor recovery in recirculation following incomplete rat brain ischemia (18-20). In rat brain mitochondria i t was shown that while recirculation normalized the depressed state 3 mitochondrial respiration after complete ischemia, i t further aggravated the aberration after incomplete ischemia. I t can be postulated that during complete ischemia the interruption of electron transfer protected the mitochondria from Piinduced insult. During incomplete ischemia, however, the presence of a trickling oxygen supply allows electron transfer to continue and therefore facilitates Pi-induced mitochondrial injury. The same explanation is applicable for substrate supply. I t has been shown that fasted animals had a significantly improved outcome following prolonged periods of ischemia and hypoxia than those that are fed or infused glucose
Vol. 37, No. i, 1985
Brain Ischemia: Mechanism of Cell Damage
73
(21). Although these observations have been attributed to the differences in the degree of acidosis between the two groups (21), i t is hypothesized that the higher degree of electron a v a i l a b i l i t y and transfer associated with a high substrate supply is responsible for the poor outcome after ischemia. Acknowledgement Supported by grant GM-29531 from the National Institute of Health. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
J.L. FARBER, K.R. CHIEN and S. MITTNACHT, JR., Am. J. Path. 102 271-281 (1981). L. MELA, Neurol. Res. 1 51-63 (1979). L. MELA, Pathoph~siology of Shock, Anoxia and Ischemia, R.A. Cowley and B F. Trump (eds.), Williams and Wilkins, Baltimore, pp. 84-95 (1982). B.K. SIESJO, J. Cereb. Blood Flow Metab. 1 155-185 (1981). B.K. SIESJO, J. Neurosurg. 60 883-908 (19~4). K.R. THULBORN, G.H. DU BONL-AY, L.W. DUCHENand G. RADDA, J. Cereb. Blood Flow Metab. 2 199-306 (1982). D.T. DELPY,R.E. GORDON, P.L. HOPE, D. PARKER, E.O.R. REYNOLDS, D. SHAW and M.D. WHITEHEAD, Pediatrics 70 310-313 (1982). S. NARUSE, Y. HORIKAMA, C. TANAKA, D. HIRAKAWA, H. NISHIKAWA, and H. WATARI, Brain Res 296 370-372 (1984). A.L. LEHNINGER, E. CA-RT~FOLI and C.S. ROSSI, Advan. Enzymol. 29 259-320 (1967). G.P. BRIERLEY, Mol. Cell. Biochem. 10 41-62 (1976). J.L.C. COELHO and A.E. VERCESI, ~-~ch. Biochem. Biophys. 204 141-147 (1980). A.C. SHEAand R.B. JENNINGS, Am J. Pathol. 67 441-452 (1972). T NAGAO, M.A. MATLIB, D. FRANKLIN, R.W. MIL'[-ARD and A. SCHWARTZ,J. Mol. Cell. Cardiol. 12 29-43 (1980). P.L. VAGHY, M.A__MATLIB and A. SCHWARTZ, Biochem. Biophys. Res. Commun. 100 37-44 (1981). F.E. HUNTER, J.F. LEVY, J. FINK, B. SCHUTZ, F. GUERRAand A. HURWITZ, J. Biol. Chem. 234 2176-2186 (1959). L.F. LANGE,T. HARTMAN and B.E. SOBEL, J. Clin. Invest. 73 1046-1052 (1984). P. CARBONEU,M. DI GENARO, R. VALLE and A.M. WEISZ, Am. J. Physiol. 240 H730-737 (1981). C-H. NORDSTROM, S. REHNCRONA and B.K. SIESJO, Acta Physiol. Scand. 97 270-272 (1976). C-H. NORDSTROM,S. REHNCRONAand B.K. SIESJO, Stroke 9 335-343 (1978). S. REHNCRONA,L. MELA and B.K. SIESJO, Stroke 10 437-~46 (1979). R.E. MYERS, Advances in Neurology. Vol. 26:---Cerebral H~poxia and Its Consequences, S. Fahn, J.N. Davis and L.P. Rowland (eds.), Raven Press, New York, pp. 195-213 (1979).
Pergamon Press
MECHANISM OF CELL DAMAGEIN BRAIN ISCHEMIA: A HYPOTHESIS K. Kariman, M.D. Box 3848, Duke University Medical Center, Durham, NC 27710 (Received in final form April 24, 1985)
Summary Mitochondria are known to develop a series of abnormalities as a result of ischemia. The i n a b i l i t y of mitochondria to resume normal function following reperfusion has been implicated as an important factor in irreversible cell damage. However, the mechanism of mitochondrial injury after ischemic brain insult is poorly understood. In this paper a hypothesis is proposed which concentrates on the interrelated roles of phosphate, calcium, and electron transport on ischemic brain cell injury. Ischemia is known to set rapidly into motion a complex series of events that involve v i r t u a l l y every organelle and subcellular system (1,2). Oxidative phosphorylation ceases, ATP stores are depleted, and v i r t u a l l y all energydependent functions cease. Although the mechanism(s) responsible for cell death in ischemic brain is poorly understood, i t is generally accepted that ischemic cell death is a consequence of mitochondrial injury (3). As recently reviewed by Siesjo (4,5) various factors (e.g. lactic acidosis, production of oxygen radicals, increased free fatty acids concentrations, etc.) have been hypothesized to be responsible for cell damage in brain ischemia. I t seems, however, that these factors are most l i k e l y the result, rather than the causes, of ischemic brain injury. In this communication a new hypothesis is proposed which focuses on the role of the triad of phosphate, calcium, and electron transport on ischemic cell injury and death in the brain. a) Role of phosphate: Dramatic increases in intracellular inorganic phosphate (Pi) in brain ischemia have been recently detected by nuclear magnetic resonance (NMR) technique in various laboratory animals (6-8). Pi, a potent agent, is known to induce mitochondrial swelling (9,10). Mitochondrial swelling is generally used as an index of mitochondrial dysfunction. I t has been demonstrated that respiration-dependent accumulation of potassium activated by Pi is responsible for the osmotic swelling of mitochondria (10). Studies carried out in the presence of external calcium indicate that Pi induces an energydissipating cycling of calcium across the inner membrane which is related to impaired oxidative phosphorylation (11). b) Role of calcium: At least in myocardial ischemia followed by reperfusion i t has been demonstrated that the resulting mitochondrial dysfunction appears to be mediated in part by calcium (12). Recently, decreased mitochondrial injury in response to inhibition of calcium transport has been demonstrated by Schwartz and associates (13). Using diltiazem, a calcium antagonist, these investigators were able to completely reverse the depression of state 3 mitochondrial respiratory rate observed following I0 minutes of coronary artery occlusion in dogs. Perhaps the most significant observation reported by this group (14) was the existence of two separable components of the Pi-induced 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
72
Brain Ischemia: Mechanism of Cell Damage
Vol. 37, No. i, 1985
mitochondriaI swelling. One was calcium antagonist ruthenium red (RR)insensitive which appears not to cause damage to mitochondria. The second component was RR-sensitive and results in inhibition and partial uncoupling of oxidative phosphorylation even in the absence of free extramitochondrial calcium. This strongly suggests the involvement of calcium in the Pi-induced damage to oxidative phosphorylation which may occur during ischemia. Furthermore, mitochondrial accumulation of calcium which is known to occur during reperfusion will further increase mitochondrial dysfunction and cellular injury (12). c) Role of electron transport: Based on observations made some 25 years ago in isolated rat liver mitochondria by Hunter et al (15), i t was clearly shown that electron transport was involved in phosphate-induced mitochondrial swelling. As proposed by these investigators, i t seems that electron transport is important for creating in the mitochondrial membrane a situation in which agents that cause swelling are able to produce their effect. Hunter et al (15) demonstrated that phosphate-induced swelling was prevented by inhibitors of electron transfer between pyridine nucleotides and oxygen as well as by the uncoupler 2,4-dinitrophenol. Using inhibitors of electron transport and 2,4-dinitrophenol in a model of perfused rat head we have observed improved mitochondrial redox responses to transient ischemic insults in situ (submitted for publication). An important observation was recently reported by Lange et al (16) in rabbit heart mitochondria. A rapid inactivation of oxidative phosphorylation was demonstrated when phosphate and oxygen were present. This inactivation was partially, but not completely, precluded by calcium antagonists. However, inactivation was prevented by incubation of mitochondria in the absence of oxygen, indicating that injury elicited by phosphate is oxygen dependent. Since intracellular Pi markedly increased with ischemia, reperfusion with oxygenated medium can paradoxically augment mitochondrial injury in this setting (17). The role of oxygen in phosphate-induced injury may be to provide an electron sink thus allowing transport into the mitochondrial matrix of phosphate and/or calcium, which could therefore be the primary mediators of injury (16). Through the same mechanism of inhibition of electron transport, anaerobiosis has been shown (9,15) to protect against v i r t u a l l y every agent known to induce mitochondrial swelling. Comments: I t is hypothesized that the triad of Pi, calcium, and electron transport is necessary for ischemic injury of the brain. The rise in intracellular Pi during ischemia, in the presence of an active electron transport, is the primary event responsible for mitochondrial edema and therefore dysfunction. Pi-induced mitochondrial injury, however, is in part mediated by calcium and calcium antagonists may have a role in preventing this phenomenon. A marked exaggeration of these phenomena occurs during reperfusion. In particular, increased mitochondrial accumulation of calcium during reperfusion results in additional cell injury. This hypothesis will explain the poor recovery in recirculation following incomplete rat brain ischemia (18-20). In rat brain mitochondria i t was shown that while recirculation normalized the depressed state 3 mitochondrial respiration after complete ischemia, i t further aggravated the aberration after incomplete ischemia. I t can be postulated that during complete ischemia the interruption of electron transfer protected the mitochondria from Piinduced insult. During incomplete ischemia, however, the presence of a trickling oxygen supply allows electron transfer to continue and therefore facilitates Pi-induced mitochondrial injury. The same explanation is applicable for substrate supply. I t has been shown that fasted animals had a significantly improved outcome following prolonged periods of ischemia and hypoxia than those that are fed or infused glucose
Vol. 37, No. i, 1985
Brain Ischemia: Mechanism of Cell Damage
73
(21). Although these observations have been attributed to the differences in the degree of acidosis between the two groups (21), i t is hypothesized that the higher degree of electron a v a i l a b i l i t y and transfer associated with a high substrate supply is responsible for the poor outcome after ischemia. Acknowledgement Supported by grant GM-29531 from the National Institute of Health. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
J.L. FARBER, K.R. CHIEN and S. MITTNACHT, JR., Am. J. Path. 102 271-281 (1981). L. MELA, Neurol. Res. 1 51-63 (1979). L. MELA, Pathoph~siology of Shock, Anoxia and Ischemia, R.A. Cowley and B F. Trump (eds.), Williams and Wilkins, Baltimore, pp. 84-95 (1982). B.K. SIESJO, J. Cereb. Blood Flow Metab. 1 155-185 (1981). B.K. SIESJO, J. Neurosurg. 60 883-908 (19~4). K.R. THULBORN, G.H. DU BONL-AY, L.W. DUCHENand G. RADDA, J. Cereb. Blood Flow Metab. 2 199-306 (1982). D.T. DELPY,R.E. GORDON, P.L. HOPE, D. PARKER, E.O.R. REYNOLDS, D. SHAW and M.D. WHITEHEAD, Pediatrics 70 310-313 (1982). S. NARUSE, Y. HORIKAMA, C. TANAKA, D. HIRAKAWA, H. NISHIKAWA, and H. WATARI, Brain Res 296 370-372 (1984). A.L. LEHNINGER, E. CA-RT~FOLI and C.S. ROSSI, Advan. Enzymol. 29 259-320 (1967). G.P. BRIERLEY, Mol. Cell. Biochem. 10 41-62 (1976). J.L.C. COELHO and A.E. VERCESI, ~-~ch. Biochem. Biophys. 204 141-147 (1980). A.C. SHEAand R.B. JENNINGS, Am J. Pathol. 67 441-452 (1972). T NAGAO, M.A. MATLIB, D. FRANKLIN, R.W. MIL'[-ARD and A. SCHWARTZ,J. Mol. Cell. Cardiol. 12 29-43 (1980). P.L. VAGHY, M.A__MATLIB and A. SCHWARTZ, Biochem. Biophys. Res. Commun. 100 37-44 (1981). F.E. HUNTER, J.F. LEVY, J. FINK, B. SCHUTZ, F. GUERRAand A. HURWITZ, J. Biol. Chem. 234 2176-2186 (1959). L.F. LANGE,T. HARTMAN and B.E. SOBEL, J. Clin. Invest. 73 1046-1052 (1984). P. CARBONEU,M. DI GENARO, R. VALLE and A.M. WEISZ, Am. J. Physiol. 240 H730-737 (1981). C-H. NORDSTROM, S. REHNCRONA and B.K. SIESJO, Acta Physiol. Scand. 97 270-272 (1976). C-H. NORDSTROM,S. REHNCRONAand B.K. SIESJO, Stroke 9 335-343 (1978). S. REHNCRONA,L. MELA and B.K. SIESJO, Stroke 10 437-~46 (1979). R.E. MYERS, Advances in Neurology. Vol. 26:---Cerebral H~poxia and Its Consequences, S. Fahn, J.N. Davis and L.P. Rowland (eds.), Raven Press, New York, pp. 195-213 (1979).