Protective effects of an adenosine deaminase inhibitor on ischemia-reperfusion injury in isolated perfused rat heart QINGYAN
ZHU,
SHANGGONG
CHEN,
AND
Division of Physiology, Cardiovascular Institute, Beijing 100037, China
ZHU,QINGYAN,SHANGGONGCHEN,ANDCUNMEIZOU. Protective effects of an adenosine deaminase inhibitor on ischemiareperfusion injury in isolatedperfused rat heart. Am. J. Physiol.
259 (Heart Circ. Physiol. 28): H835-H838, 1990.-We tested the effect of the adenosinedeaminaseinhibitor erythro-9-(2hydroxy-3-nonyl)adenine (EHNA) on ischemia-reperfusioninjury in isolated perfusedrat heart. In the ischemia-reperfusion group (n = IO), ventricular fibrillation occurred within 3 min of reperfusion after the 4O-min ischemicperiod. The incidence of ventricular fibrillation was 90% with a mean duration of 3.15 t 0.97 (SE) min. Resting tension increasedsignificantly. By contrast, the incidence of ventricular fibrillation after reperfusion in the EHNA-treated (5 PM) group (n = 10) was20% (P < O.Ol), and the duration was 0.30 t 0.21 min (P < 0.01). Resting tension was significantly lower and around the normal level in the EHNA-treated group (P < 0.01). Contraction amplitude and heart rate recoveredto nearly normal compared with the ischemia-reperfusiongroup (P < 0.01). Coronary flow was greater in the EHNA-treated group (P < 0.01). It is concludedthat EHNA protects the heart, possibly by accumulation of adenosinethat benefits the hearts and by blocking the xanthine oxidasepathway for free radical generation. oxygen free radicals; ventricular fibrillation; contraction amplitude; heart rate
that ATP is degraded to AMP, adenosine, inosine, and hypoxanthine successively during ischemia (15, 17). This will result in the loss of ATP, adenosine, and other adenine nucleotides from myocytes and accumulation of hypoxanthine that is the substrate of a superoxide anion (O,*), generating a reaction that is catalyzed by xanthine oxidase (8, 17). Hence, ischemia-reperfusion will induce functional and structural damage by loss of energy and by generation of oxygen-derived free radicals. Erythro-9-(2-hydroxy-3nonyl)adenine (EHNA) is an adenosine deaminase inhibitor that can block the deamination of adenosine to inosine. We hypothesize that EHNA would be able to increase the level of endogenous adenosine that is a protector of ischemic-reperfused myocardium (3, 14, 20) and decrease the generation of 02. by reducing the concentration of hypoxanthine. Therefore, EHNA would have a protective effect on the myocardium. This hypothesis was tested in the present study. IT HAS BEEN DEMONSTRATED
MATERIALS
AND METHODS
Isolated rat heart perfusion and ischemia-reperfusion model. Male Wistar rats weighing X0-250 g were anes0363-6135/90
$1.50
Copyright
CUNMEI
ZOU
Chinese Academy of Medical Sciences,
thetized with 20% urethan (1 g/kg ip), and heparin sodium (200 U) was administered intravenously. The chest was opened, and the heart was excised and perfused by the method of Langendorff (24). The perfusate had the following composition (in mM): 118 NaCl, 4.75 KCl, 2.52 CaC12, 1.19 KH2P04, 1.19 MgCl,, 25 NaHC03, and 10.1 glucose. The pH was 7.35-7.45 at 36”C, and the Pop was 500-600 mmHg after gassing with 95% 02-5% COZ. Perfusion pressure was kept constant at 75 cmHzO. After 15 min of equilibration, the hearts were divided randomly into three groups: 1) control (n = 9), aerobic perfusion for 50 min; 2) ischemia-reperfusion (n = lo), global ischemia (36°C) 40 min followed by aerobic reperfusion for 10 min; 3) EHNA (n = lo), same as the ischemia-reperfusion group but with 5 PM EHNA perfusion 10 min before the onset of ischemia. During the experimental period, coronary flow, contraction amplitude, resting tension, and heart rate were recorded. During reperfusion, the incidence and duration of ventricular fibrillation were also monitored. Statistical analysis. Measurement data in various groups were means & SE. Significant differences (P c 0.05) were determined with nonpaired Student’s t test. Enumeration data (incidence of ventricular fibrillation) were determined for significant differences (P < 0.05) with the rate test for two samples. RESULTS
In the control group, the coronary flow, contraction amplitude, resting tension, and heart rate were constant during the 50 min of aerobic perfusion period (Figs. l4) .
In the ischemia-reperfusion group, contraction amplitude and heart rate decreased after the onset of ischemia and almost reached zero after 15 min of ischemia. The resting tension also decreased in the first 20 min of ischemia but increased progressively thereafter. Ventricular fibrillation developed in 90% of the ischemic hearts and occurred within the first 3 min of reperfusion. The duration of ventricular fibrillation was 3.15 t 0.97 min. With reperfusion, the resting tension increased further, and contraction amplitude and heart rate recovered slightly but remained well below the control level (Figs. 1- 4) In’ the EHNA group, the coronary flow, contraction amplitude, and heart rate in the ischemic period were similar to those of the ischemia-reperfusion group, but
0 1990 the American
Physiological
Society
H835
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on November 7, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
H836
EHNA
INHIBITION
AND
10
,
ISCHEMIA-REPERFUSION
INJURY
EHNA
isc Con
\
80
-Rep
Isc -Rep
60 n
8
40-
--4 ** *
EHNA Con
0 0
5
10
15
20
lschemia
25
30
35
40
45
50
0
FIG. 1. Effects of EHNA on coronary flow in isolated perfused rat heart. o--+ control (Con) group (aerobic perfusion for 50 min). xx, ischemia-reperfusion (Isc-rep) group (40 min ischemia followed by 10 min reperfusion). 0- - - - - -0, EHNA group (same as ischemia-reperfusion group but 5 FM EHNA perfusion 10 min before ischemia and during whole reperfusion period). Each point and bars represent means t SE. Compared with ischemia-reperfusion group, * P < 0.05, ** P
5 PM, has a strong inhibitory effect on adenosine deaminase (1, 22), and it can increase adenosine levels in isolated rat hearts (1). Adenosine is a powerful vasodilator. It is understandable that accumulation of adenosine in ischemic myocardium would greatly enhance the coronary flow during the reperfusion period and thereby increase oxygen supply to the myocardium. Adenosine also inhibits conduction through the atrioventricular node and depresses sinoatrial node excitation (4). These dromotropic and chronotropic effects might be responsible for the inhibition of the occurrence of ventricular fibrillation. In addition, adenosine has been reported (9) to increase myocardial prostacyclin production, which can markedly reduce the
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on November 7, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
EHNA
INHIBITION
AND
ISCHEMIA-REPERFUSION
incidence of ventricular fibrillation after reperfusion (10). Accumulated adenosine can also serve as an ATP precursor to replenish ATP stores (3, 20) and thereby enhance recovery of contractile function after reperfusion in the EHNA-treated group. These results are in agreement with those of others (3, 14, 20) in that adenosine can improve regional myocardial blood flow and function, inhibit arrhythmia, and preserve the function and structure of endothelial cells and high-energy phosphate. Adenosine is also reported to inhibit granulocyte infiltration (3) and 0,. generation by activated neutrophils (13). But in our experiments, neutrophils were absent (saline-perfused heart) so this mechanism cannot be involved. Some reports indicate that (2, 18, 25) in the ischemicreperfused rat heart, the production of oxygen free radicals occurs mainly via the xanthine oxidase pathway. In brief, during ischemia, ATP is degraded to AMP, adenosine, inosine, and hypoxanthine, successively, and xanthine dehydrogenase is converted to xanthine oxidase. Reperfusion provides the ischemic myocardium with the molecular oxygen that acts as an electron acceptor during the conversion of hypoxanthine to xanthine catalyzed by xanthine oxidase. In this reaction, 0,. is produced (8, 17). EHNA, an inhibitor of adenosine deaminase, would block the conversion of adenosine to inosine and consequently decrease the amount of hypoxanthine production and the resultant 0~. in ischemic-reperfused myocardium. Oxygen-derived free radicals (0,. , .OH, etc.) are generally considered a very important factor responsible for ischemia-reperfusion injury (17). Hence, EHNA may have a protective effect on ischemia-reperfusion injury in rat heart by decreasing 0;. generation. EHNA was also found to be inhibitory to a variety of biological functions. For example, it has been reported to inhibit viral replication (19), to potentiate the inhibition of lymphocyte-mediated cytolysis (22), to inhibit motility of sea urchin sperm (5), and even to scavenge 0,. directly at high concentration (1 pmol/ml) (15). Thus it is possible that EHNA may have protective effects on ischemia-reperfusion injury other than as an adenosine deaminase inhibitor, even at the concentration we used (5 PM). In conclusion, the adenosine deaminase inhibitor EHNA can dramatically protect the ischemic-reperfused isolated rat heart possibly by preserving adenosine and blocking the xanthine oxidase pathway for free radical generation. EHNA appears to be a useful agent in the protection of ischemic-reperfused myocardium. We are EHNA we Address of Virginia Received
very grateful to Dr. Robert M. Berne for sending us the used in this research. for reprint requests: Q. Zhu, Dept. of Physiology, University Health Sciences Center, Charlottesville, VA 22908.
7 August
1989; accepted
in final
form
3 May
1990.
3.
5.
PARMLEY, D. M. YELLON, AND J. M. DOWNEY. Infarct size limitation by the xanthine oxidase inhibitor, allopurinol, in closedchest dogs with small infarcts. Cardiovasc. Res. 19: 686-692, 1985. BABBITT, D. G., R. VIRMANI, AND M. B. FORMAN. Intracoronary adenosine administered after reperfusion limits vascular injury after prolonged ischemia in the canine model. Circulation 80: 1388effects of adenosine. In: Adenosine: Receptors and Modulation of Cell Function, edited by V. Stefanovich, K. Rudolphi, and P. Schubert. Oxford, UK: IRL, 1985. BOUCHARD, P., S. M. PENNINGGROTH, A. CHEUNG, C. GAGNON, AND C. W. BARDIN. Erythro-9-[3-(2-hydroxynonyl)]adenine is an inhibitor of sperm motility that blocks dynein ATPase and protein carboxylmethylase activities. Proc. Natl. Acad. Sci. USA 78: 1033-
1036, 1981. 6. BRADFORD, M. M. A rapid and sensitive 7. 8.
9. 10.
11.
method for the quantitation of microgram quantities for protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. CAMPBELL, T. J. A possible role for free radicals in cardiac reperfusion phenomena. Aust. NZ J. Med. 17: 459-460, 1987. CHAMBERS, D. E., D. A. PARKS, G. PATTERSON, R. ROY, J. M. MCCORD, S. YOSHIDA, L. F. PARMLEY, AND J. M. DOWNEY. Xanthine oxidase as a source of free radical damage in myocardial ischemia. J. Mol. Cell. Cardiol. 17: 145-152, 1985. CIABATTONI, G., AND A. WENNMALM. Adenosine induced coronary release of prostacycline at normal and low pH in isolated heart of rabbit. Br. J. Pharmacol. 85: 557-563, 1985. COKER, S. J., AND J. R. PARRATT. The effects of nafazatrom on arrhythmias and prostanoid release during coronary artery occlusion and reperfusion in anaesthetised greyhounds. J. Mol. Cell. Cardiol. 16: 43-52, 1984. CRONSTEIN, B. N., E. D. ROSENSTEIN, S. B. KRAMER, G. WEISSMANN, AND R. HIRSCHHORN. Adenosine: a physiologic modulator of superoxide anion generation by human neutrophils. Adenosine acts via an A, receptor on human neutrophils. J. Immunol. 135:
1366-1371,1985. 12. DOWNEY, J. M.,
13. 14.
15. 16. 17. 18.
19.
20.
1. ACHTERBERG, 2.
H837
1399, 1989. 4. BERNE, R. M. Some cardiovascular
REFERENCES P. W., AND J. W. DE JONG. Adenosine deaminase inhibition and myocardial adenosine metabolism during &hernia. Adv. Myocardiol. 6: 465-472, 1985. AKIZUKI, S., S. YOSHIDA, D. E. CHAMBERS, L. J. EDDY, L. F.
INJURY
21.
T. MIURA, L. J. EDDY, D. E. CHAMBERS, T. MELLERT, D. J. HEARSE, AND D. M. YELLON. Xanthine oxidase is not a source of free radicals in the ischemic rabbit heart. J. Mol. Cell. Cardiol. 19: 1053-1060, 1987. ENGLER, R. Consequences of activation and adenosine-mediated inhibition of granulocytes during myocardial ischemia. Federation Proc. 46: 2407-2412, 1987. GRUBER, H. E., M. E. HOFFER, D. R. MCALLISTER, P. K. LAIKIND, T. A. LANE, G. W. SCHMID-SCHOENBEIN, AND R. L. ENGLER. Increased adenosine concentration in blood from ischemic myocardium by AICA riboside. Effects on flow, granulocytes, and injury. Circulation 80: 1400-1411, 1989. KOKE, J. R., L. M. Fu, D. SUN, D. M. VAUGHAN, AND N. BITTAR. Inhibitors of adenosine catabolism improve recovery of dog myocardium after ischemia. Mol. Cell. Biochem. 86: 107-113, 1989. KOSTER, J. F., P. BIEMOND, AND H. STAM. Lipid peroxidation and myocardial ischemia damage: cause or consequence? Basic Res. Cardiol. 82, Suppl. 1: 253-260, 1987. MCCORD, J. M. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312: 159-163, 1985. MYERS, C. L., S. J. WEISS, M. M. KIRSCH, AND M. SHLAFER. Involvement of hydrogen peroxide and hydroxyl radical in the “oxygen paradox”: reduction of CK release by catalase, allopurinol or deferoxamaine, but not by superoxide dismutase. J. Mol. Cell. Cardiol. 17: 675-684, 1985. NORTH, T. W., AND S. S. COHEN. Erythro-9-(2-hydroxy-3nonyl)adenine as a specific inhibitor of herpes simplex virus replication in the presence and absence of adenosine analogues. Proc. Natl. Acad. Sci. USA 75: 4684-4688, 1978. OLAFSSON, B., M. B. FORMAN, D. W. PUETT, A. Pou, C. U. CATES, G. C. FRIESINGER, AND R. VIRMANI. Reduction of reperfusion injury in the canine preparation by intracoronary adenosine: importance of the endothelium and the no-reflow phenomenon. Circulation 76: 1135-1145, 1987. SCHMID-SCHONBEIN, G. W. Capillary plugging by granulocytes and the no-reflow phenomenon in the microcirculation. Federation Proc. 46: 2397-2401, 1987.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on November 7, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
H838 22. WOLBERG, aminase
EHNA
G., AND T. P. ZIMMERMAN. inhibitors on lymphocyte-mediated
INHIBITION Effects
Acad. Sci. 451: 215-226,19&L 23. WV, S., L. Fu, J. R. KOKE, AND N. BITTAR.
AND
of adenosine cytolysis. Ann.
ISCHEMIA-REPERFUSION de-
NY
Contractility, ATP, and creatine phosphate during myocardial ischemia and reperfusion: the effects of adenosine and inhibition of adenosine catabolism in the dog heart. Cytobios 50: 7-12, 1987.
INJURY
24. ZHU, Q., AND S. CHEN. Effects of verapamil on ouabain intoxication to guinea-pig heart. Chin. Circ. J. 2: 422-424, 1987 (English abstract). 25. ZHU, Q., S. CHEN, B. ZHAO, W. XIN, C. Zou, AND J. HAO. ESR detection of oxygen free radicals generated in ischemic-reperfused rat heart and the protective effects of SOD and DTZ (Abstract). Chin. J. CardioZ. 17: 304, 1989.
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on November 7, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
ZHU,
SHANGGONG
CHEN,
AND
Division of Physiology, Cardiovascular Institute, Beijing 100037, China
ZHU,QINGYAN,SHANGGONGCHEN,ANDCUNMEIZOU. Protective effects of an adenosine deaminase inhibitor on ischemiareperfusion injury in isolatedperfused rat heart. Am. J. Physiol.
259 (Heart Circ. Physiol. 28): H835-H838, 1990.-We tested the effect of the adenosinedeaminaseinhibitor erythro-9-(2hydroxy-3-nonyl)adenine (EHNA) on ischemia-reperfusioninjury in isolated perfusedrat heart. In the ischemia-reperfusion group (n = IO), ventricular fibrillation occurred within 3 min of reperfusion after the 4O-min ischemicperiod. The incidence of ventricular fibrillation was 90% with a mean duration of 3.15 t 0.97 (SE) min. Resting tension increasedsignificantly. By contrast, the incidence of ventricular fibrillation after reperfusion in the EHNA-treated (5 PM) group (n = 10) was20% (P < O.Ol), and the duration was 0.30 t 0.21 min (P < 0.01). Resting tension was significantly lower and around the normal level in the EHNA-treated group (P < 0.01). Contraction amplitude and heart rate recoveredto nearly normal compared with the ischemia-reperfusiongroup (P < 0.01). Coronary flow was greater in the EHNA-treated group (P < 0.01). It is concludedthat EHNA protects the heart, possibly by accumulation of adenosinethat benefits the hearts and by blocking the xanthine oxidasepathway for free radical generation. oxygen free radicals; ventricular fibrillation; contraction amplitude; heart rate
that ATP is degraded to AMP, adenosine, inosine, and hypoxanthine successively during ischemia (15, 17). This will result in the loss of ATP, adenosine, and other adenine nucleotides from myocytes and accumulation of hypoxanthine that is the substrate of a superoxide anion (O,*), generating a reaction that is catalyzed by xanthine oxidase (8, 17). Hence, ischemia-reperfusion will induce functional and structural damage by loss of energy and by generation of oxygen-derived free radicals. Erythro-9-(2-hydroxy-3nonyl)adenine (EHNA) is an adenosine deaminase inhibitor that can block the deamination of adenosine to inosine. We hypothesize that EHNA would be able to increase the level of endogenous adenosine that is a protector of ischemic-reperfused myocardium (3, 14, 20) and decrease the generation of 02. by reducing the concentration of hypoxanthine. Therefore, EHNA would have a protective effect on the myocardium. This hypothesis was tested in the present study. IT HAS BEEN DEMONSTRATED
MATERIALS
AND METHODS
Isolated rat heart perfusion and ischemia-reperfusion model. Male Wistar rats weighing X0-250 g were anes0363-6135/90
$1.50
Copyright
CUNMEI
ZOU
Chinese Academy of Medical Sciences,
thetized with 20% urethan (1 g/kg ip), and heparin sodium (200 U) was administered intravenously. The chest was opened, and the heart was excised and perfused by the method of Langendorff (24). The perfusate had the following composition (in mM): 118 NaCl, 4.75 KCl, 2.52 CaC12, 1.19 KH2P04, 1.19 MgCl,, 25 NaHC03, and 10.1 glucose. The pH was 7.35-7.45 at 36”C, and the Pop was 500-600 mmHg after gassing with 95% 02-5% COZ. Perfusion pressure was kept constant at 75 cmHzO. After 15 min of equilibration, the hearts were divided randomly into three groups: 1) control (n = 9), aerobic perfusion for 50 min; 2) ischemia-reperfusion (n = lo), global ischemia (36°C) 40 min followed by aerobic reperfusion for 10 min; 3) EHNA (n = lo), same as the ischemia-reperfusion group but with 5 PM EHNA perfusion 10 min before the onset of ischemia. During the experimental period, coronary flow, contraction amplitude, resting tension, and heart rate were recorded. During reperfusion, the incidence and duration of ventricular fibrillation were also monitored. Statistical analysis. Measurement data in various groups were means & SE. Significant differences (P c 0.05) were determined with nonpaired Student’s t test. Enumeration data (incidence of ventricular fibrillation) were determined for significant differences (P < 0.05) with the rate test for two samples. RESULTS
In the control group, the coronary flow, contraction amplitude, resting tension, and heart rate were constant during the 50 min of aerobic perfusion period (Figs. l4) .
In the ischemia-reperfusion group, contraction amplitude and heart rate decreased after the onset of ischemia and almost reached zero after 15 min of ischemia. The resting tension also decreased in the first 20 min of ischemia but increased progressively thereafter. Ventricular fibrillation developed in 90% of the ischemic hearts and occurred within the first 3 min of reperfusion. The duration of ventricular fibrillation was 3.15 t 0.97 min. With reperfusion, the resting tension increased further, and contraction amplitude and heart rate recovered slightly but remained well below the control level (Figs. 1- 4) In’ the EHNA group, the coronary flow, contraction amplitude, and heart rate in the ischemic period were similar to those of the ischemia-reperfusion group, but
0 1990 the American
Physiological
Society
H835
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on November 7, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
H836
EHNA
INHIBITION
AND
10
,
ISCHEMIA-REPERFUSION
INJURY
EHNA
isc Con
\
80
-Rep
Isc -Rep
60 n
8
40-
--4 ** *
EHNA Con
0 0
5
10
15
20
lschemia
25
30
35
40
45
50
0
FIG. 1. Effects of EHNA on coronary flow in isolated perfused rat heart. o--+ control (Con) group (aerobic perfusion for 50 min). xx, ischemia-reperfusion (Isc-rep) group (40 min ischemia followed by 10 min reperfusion). 0- - - - - -0, EHNA group (same as ischemia-reperfusion group but 5 FM EHNA perfusion 10 min before ischemia and during whole reperfusion period). Each point and bars represent means t SE. Compared with ischemia-reperfusion group, * P < 0.05, ** P
5 PM, has a strong inhibitory effect on adenosine deaminase (1, 22), and it can increase adenosine levels in isolated rat hearts (1). Adenosine is a powerful vasodilator. It is understandable that accumulation of adenosine in ischemic myocardium would greatly enhance the coronary flow during the reperfusion period and thereby increase oxygen supply to the myocardium. Adenosine also inhibits conduction through the atrioventricular node and depresses sinoatrial node excitation (4). These dromotropic and chronotropic effects might be responsible for the inhibition of the occurrence of ventricular fibrillation. In addition, adenosine has been reported (9) to increase myocardial prostacyclin production, which can markedly reduce the
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.108.009.184) on November 7, 2018. Copyright © 1990 American Physiological Society. All rights reserved.
EHNA
INHIBITION
AND
ISCHEMIA-REPERFUSION
incidence of ventricular fibrillation after reperfusion (10). Accumulated adenosine can also serve as an ATP precursor to replenish ATP stores (3, 20) and thereby enhance recovery of contractile function after reperfusion in the EHNA-treated group. These results are in agreement with those of others (3, 14, 20) in that adenosine can improve regional myocardial blood flow and function, inhibit arrhythmia, and preserve the function and structure of endothelial cells and high-energy phosphate. Adenosine is also reported to inhibit granulocyte infiltration (3) and 0,. generation by activated neutrophils (13). But in our experiments, neutrophils were absent (saline-perfused heart) so this mechanism cannot be involved. Some reports indicate that (2, 18, 25) in the ischemicreperfused rat heart, the production of oxygen free radicals occurs mainly via the xanthine oxidase pathway. In brief, during ischemia, ATP is degraded to AMP, adenosine, inosine, and hypoxanthine, successively, and xanthine dehydrogenase is converted to xanthine oxidase. Reperfusion provides the ischemic myocardium with the molecular oxygen that acts as an electron acceptor during the conversion of hypoxanthine to xanthine catalyzed by xanthine oxidase. In this reaction, 0,. is produced (8, 17). EHNA, an inhibitor of adenosine deaminase, would block the conversion of adenosine to inosine and consequently decrease the amount of hypoxanthine production and the resultant 0~. in ischemic-reperfused myocardium. Oxygen-derived free radicals (0,. , .OH, etc.) are generally considered a very important factor responsible for ischemia-reperfusion injury (17). Hence, EHNA may have a protective effect on ischemia-reperfusion injury in rat heart by decreasing 0;. generation. EHNA was also found to be inhibitory to a variety of biological functions. For example, it has been reported to inhibit viral replication (19), to potentiate the inhibition of lymphocyte-mediated cytolysis (22), to inhibit motility of sea urchin sperm (5), and even to scavenge 0,. directly at high concentration (1 pmol/ml) (15). Thus it is possible that EHNA may have protective effects on ischemia-reperfusion injury other than as an adenosine deaminase inhibitor, even at the concentration we used (5 PM). In conclusion, the adenosine deaminase inhibitor EHNA can dramatically protect the ischemic-reperfused isolated rat heart possibly by preserving adenosine and blocking the xanthine oxidase pathway for free radical generation. EHNA appears to be a useful agent in the protection of ischemic-reperfused myocardium. We are EHNA we Address of Virginia Received
very grateful to Dr. Robert M. Berne for sending us the used in this research. for reprint requests: Q. Zhu, Dept. of Physiology, University Health Sciences Center, Charlottesville, VA 22908.
7 August
1989; accepted
in final
form
3 May
1990.
3.
5.
PARMLEY, D. M. YELLON, AND J. M. DOWNEY. Infarct size limitation by the xanthine oxidase inhibitor, allopurinol, in closedchest dogs with small infarcts. Cardiovasc. Res. 19: 686-692, 1985. BABBITT, D. G., R. VIRMANI, AND M. B. FORMAN. Intracoronary adenosine administered after reperfusion limits vascular injury after prolonged ischemia in the canine model. Circulation 80: 1388effects of adenosine. In: Adenosine: Receptors and Modulation of Cell Function, edited by V. Stefanovich, K. Rudolphi, and P. Schubert. Oxford, UK: IRL, 1985. BOUCHARD, P., S. M. PENNINGGROTH, A. CHEUNG, C. GAGNON, AND C. W. BARDIN. Erythro-9-[3-(2-hydroxynonyl)]adenine is an inhibitor of sperm motility that blocks dynein ATPase and protein carboxylmethylase activities. Proc. Natl. Acad. Sci. USA 78: 1033-
1036, 1981. 6. BRADFORD, M. M. A rapid and sensitive 7. 8.
9. 10.
11.
method for the quantitation of microgram quantities for protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. CAMPBELL, T. J. A possible role for free radicals in cardiac reperfusion phenomena. Aust. NZ J. Med. 17: 459-460, 1987. CHAMBERS, D. E., D. A. PARKS, G. PATTERSON, R. ROY, J. M. MCCORD, S. YOSHIDA, L. F. PARMLEY, AND J. M. DOWNEY. Xanthine oxidase as a source of free radical damage in myocardial ischemia. J. Mol. Cell. Cardiol. 17: 145-152, 1985. CIABATTONI, G., AND A. WENNMALM. Adenosine induced coronary release of prostacycline at normal and low pH in isolated heart of rabbit. Br. J. Pharmacol. 85: 557-563, 1985. COKER, S. J., AND J. R. PARRATT. The effects of nafazatrom on arrhythmias and prostanoid release during coronary artery occlusion and reperfusion in anaesthetised greyhounds. J. Mol. Cell. Cardiol. 16: 43-52, 1984. CRONSTEIN, B. N., E. D. ROSENSTEIN, S. B. KRAMER, G. WEISSMANN, AND R. HIRSCHHORN. Adenosine: a physiologic modulator of superoxide anion generation by human neutrophils. Adenosine acts via an A, receptor on human neutrophils. J. Immunol. 135:
1366-1371,1985. 12. DOWNEY, J. M.,
13. 14.
15. 16. 17. 18.
19.
20.
1. ACHTERBERG, 2.
H837
1399, 1989. 4. BERNE, R. M. Some cardiovascular
REFERENCES P. W., AND J. W. DE JONG. Adenosine deaminase inhibition and myocardial adenosine metabolism during &hernia. Adv. Myocardiol. 6: 465-472, 1985. AKIZUKI, S., S. YOSHIDA, D. E. CHAMBERS, L. J. EDDY, L. F.
INJURY
21.
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