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Protective Effect of Diltiazem on Myocardial Ischemic Injury Associated With ·OH Generation
Comp. Biochem. Physiol. Vol. 117A, No. 2, pp. 257–261, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00262-9
Protective Effect of Diltiazem on Myocardial Ischemic Injury Associated With ⋅OH Generation Toshio Obata and Yasumitsu Yamanaka Department of Pharmacology Oita Medical University, Hasama-machi, Oita 879-55, Japan ABSTRACT. We examined the protective effect of diltiazem, a calcium antagonist, on myocardial ischemic injury associated with generation of hydroxyl free radicals (⋅OH). Salicylic acid in Ringer’s solution (0.5 nmol ⋅ µl 21 ⋅ min21 ) was infused directly through a microdialysis probe to detect the generation of ⋅OH as reflected by the formation of 2,3-dihydroxybenzoic acid (DHBA) in the myocardium. Cardiac dialysate was assayed for 2,3-DHBA by a high-performance liquid chromatographic-electrochemical (HPLC-EC) procedure. The heart was subjected to myocardial ischemia for 15 min by occlusion of left anterior descending artery (LAD). The presence of ⋅OH was indicated in the ischemic reperfused rat heart. However, when heart was reperfused, the elevation of 2,3-DHBA by 15-min ischemia was not observed in the ischemic zone following systemic administration of diltiazem (100 µg ⋅ min21 ⋅ kg21 ), a calcium antagonist. When corresponding experiments were performed with allopurinol (10 mg ⋅ kg21 ) administration of i.v. injection, the elevation of 2,3-DHBA was not observed. These results suggest that diltiazem may suppress the ⋅OH generation from xanthine-xanthine oxidase system by ischemia-reperfusion. comp biochem physiol 117A;2:257–261, 1997. 1997 Elsevier Science Inc. KEY WORDS. Allopurinol, calcium overload, diltiazem, hydroxyl free radical, ischemia, microdialysis, salicylic acid
INTRODUCTION The deleterious effects of reperfusion on the ischemic myocardium have been linked to the production of oxygenderived free radicals (9,13,16). Recent studies (10,16,21) suggest an involvement of oxygen free radicals in the pathophysiology of myocardial ischemia–induced cell damage. Intracellular calcium level builds and catalyzes the conversion of xanthine dehydrogenase to xanthine oxidase (26), which produces superoxide. Calcium (Ca21 ) ion may be a very important factor in the induction of irreversible ischemic injury (25). However, the interaction between intracellular Ca21 overload and oxygen free radicals in myocardium is not clear. Intracellular Ca21 level has been proposed as a source of oxidative toxicity (2). Superoxide, either directly or after conversion to hydroxyl radical (4,12), damages biological membrane (17) and other cellular components, including DNA (1), resulting in cell death. The hydroxyl free radical (⋅OH) reacts with salicylate and generates 2,3- and 2,5-dihydroxybenzoic acid (DHBA), which can be measured electrochemically in picomole quantity by high pressure liquid chromatography (HPLC) (5,24). However, 2,3-DHBA can be non-enzymatically Address reprint requests to: Toshio Obata, Department of Pharmacology, Oita Medical University, Hasama-machi, Oita 879-55, Japan. Tel. 097586-5724; Fax 0975-86-5729. Abbreviations—HPLC, high-performance liquid chromatography; 2,3DHBA, 2,3-dihydroxybenzoic acid; ⋅OH, hydroxyl free radical.
formed by the aromatic hydroxylation of ⋅OH and can provide an assay for ⋅OH formation (5,8,24). The present study focused on a possible use of salicylate hydroxylation as an in vivo trapping procedure (3,5,8,23,24) for monitoring the time course of 2,3-DHBA generation in the heart. The present study attempted to examine the protective effect of diltiazem, a calcium antagonist, on myocardial ischemic/reperfusion injury caused by ⋅OH generation. MATERIALS AND METHODS Animals Male Wistar rats weighing 350–450 g were anesthetized with chloral hydrate (400 mg ⋅ kg21 i.p.). The level anesthesia was maintained with continuous intravenous infusion of chloral hydrate (20 mg ⋅ kg21 ⋅ h21 ). Artificial ventilation was maintained with a constant-volume respiration using room air mixed with oxygen. The heart rate, arterial blood pressure, and electrocardiogram (ECG) were monitored and recorded continuously. Heparin sodium (200 U/kg) was administered intravenously before probe implantation; 100 U/ kg was then given every 1 hr to prevent blood coagulation. Diltiazem (Herbesser; Tanabe Seiyaku Co., Ltd., Tokyo, Japan; 100 µg ⋅ min21 ⋅ kg21 ) was continuously administered by intravenous infusion. Changes in heart rate were monitored by minipolygraph (Nihon Kohden, RM-6100, Tokyo, Japan). This study was approved by the Ethical Committee for Animal Experiments, Oita Medical University.
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Cardiac Microdialysis We used an in vivo microdialysis technique (20) to examine the ⋅OH generation on myocardial ischemia/reperfusion injury. With the animal in lateral position, the left fifth or sixth rib on the left side was partially removed to observe the heart. A small incision was made in the pericardium, and, with a fine guiding needle. The dialysis probe was implanted into the ischemic zone of the left anterior descending coronary artery (LAD). The inlet side of the microdialysis probe was connected by a polyethylene tube to a 1-ml syringe, which was driven by a microinjection pump (Carnegie Medicine CMA/100, Sweden). The outlet side of the probe was connected to the same tube and placed into a small collecting tube. Dialysis probe was perfused with Ringer’s solution containing 147 mM NaCl, 2,3 mM CaCl2, 4 mM KCl, pH 7.0. To determine an adequate perfusion speed, we tested in vitro to calculate the recovery of 2,3-DHBA through the membrane under various conditions. The dialysis probe was bathed in 37°C Ringer’s solution with a constant 2,3-DHBA concentration. The dialysate 2,3-DHBA concentration was measured directly by HPLC with electrochemical (EC) procedure. When the probe (3-mm exposure) was perfused with 1 µl ⋅ min21 in vitro, the relative recovery rate of 1 µM 2,3-DHBA was an average of about 10%. Using this perfusion speed in vitro, we obtained uniform relative recovery rates with different 2,3-DHBA concentrations in the testing dialysate solution. On the basis of these results, we chose a perfusion speed of 1 µ21 ⋅ min21 for in vivo experiments. Ringer’s solution containing 0.5 mM salicylic acid (0.5 n mol ⋅ µl21 ⋅ min21 ) was infused directly through the microdialysis probe in the ventricular LAD. The dialysis samples from the myocardium were immediately injected for analysis into an HPLCEC system equipped with a glassy carbon working electrode (EICOM, Kyoto, Japan) and reverse-phase column, an Eicompak MA-50DS column (5 µm, 4,6 3 150 mm, Eicom). The working electrode was set at a detector potential of 0.75 V. Each liter of mobile phase contained 1.5 g heptane sulfonic acid sodium salt (Sigma Chemical, St Louis, MO), 0.1 g Na2 EDTA, 3 ml triethylamine (Wako Pure Chemical Industries, Osaka, Japan) and 125 ml acetonitrile (Wako) dissolved in H2O. The pH of solution was adjusted to 2.8 with 3 ml phosphoric acid (Wako) and filtered through a Millipore filter before the addition of acetonitrile. Flow rate of 0.7 and 0.9 ml ⋅ min21 allowed the sample to be assayed. The dialysate (1 µl ⋅ min21 ) was then collected every 15 min into small collecting tubes containing 15 µl of ice-cold 0.1 N HClO4 and assayed immediately for 2,3DHBA by a HPLC-EC procedure.
tube from the surrounding coronary artery. The heart was subjected to regional ischemia for 15 min by the occlusion of LAD followed by reperfusion. Statistical Methods The results were expressed as the mean SEM of the 2,3DHBA outputs. Statistical analysis were performed by ANOVA test. A P value of ,0.05 was considered significant. RESULTS After a 60-min washout with Ringer’s solution, we determined the time course of norepinephrine levels in a dialysates collected at 15-min intervals over a period of 135 min (Fig. 1). When the dialysate norepinephrine level had reached an almost steady-state level, the heart was subjected to regional ischemia for 15 min by the occlusion of LAD, followed then by reperfusion. It reached a low steady-state level of 0.148 6 0.015 nmol/ml at 150–165 min after probe implantation. When occlusion was begun and reperfusion was started, we confirmed typical changes in ECG in control rats. The incidence of ischemic arrhythmia was observed at 13 sec after occlusion of the coronary artery. Ventricular tachycardia and ventricular fibrillation occurred at 21 sec by reperfusion. Ischemia decreased heart rate and mean arterial pressure. Heart rate decreased from 348 6 25 beats ⋅ min 21 during ischemia to 306 6 27 beats ⋅ min21 after reperfusion, but this change was not significant. Mean arterial pressure significantly decreased from 79 6 14 mmHg during ischemia to 67 6 19 mmHg after reperfusion. After systemic administration of diltiazem (100 µg ⋅ min 21 ⋅ kg21 ) starting
FIG. 1. Time course of dialysate norepinephrine (NE) levels
Preparation of Ischemic Rats After the microdialysis probe implantation in ischemic zone, the LAD branch was clamped by a thread through a
in extracellular fluid of myocardium. Dialysate NE gradually decreased over the first 150 min and then reached low steady levels. Values are expressed as mean 6 SE of six rats. *p , 0.01 significant change.
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FIG. 2. Typical changes in the electrocardiogram (ECG). Left anterior descending (LAD) branch was clamped by thread through a tube from surrounding coronary artery. When the heart was subjected to 15 min of regional ischemia by LAD occlusion, incidence of ischemic and reperfusion arrhythmia were observed (Control). After systemic administration of diltiazem (100 mg ⋅ min21 ⋅ kg21 ), incidence of ischemic arrhythmia was not observed in ischemic reperfused heart (Diltiazem). When corresponding experiments were performed with allopurinol (10 mg ⋅ kg21 ) administration of i.v. injection, the incidence of ischemic arrhythmia was not observed (Allopurinol).
30 min (120 min after experiment), incidence of ischemic arrhythmia was not observed in the ischemic-reperfused heart. When corresponding experiments were performed with allopurinol (10 mg ⋅ kg21) administration of i.v. injection, the incidence of ischemic arrhythmia was not observed (Fig. 2). However, the systemic administration of diltiazem gradually decreased heart rate (Fig. 3). Reperfusion gradually decreased heart rate (from 310 6 28 to 292 6 11 beats ⋅ min21 ) or mean arterial pressure (from 68 6 21 to 65 6 18 mmHg).
Effect of Diltiazem and Allopurinol on ⋅OH Formation in Ischemic-reperfused Rat Heart The presence of ⋅OH was observed in ischemic-reperfused rat heart. Sodium salicylate was infused for 150 min to trap ⋅OH, which was formed by ischemia-reperfusion of the myocardium. The authentic standards of 2,3-DHBA (reaction products of salicylic acid and ⋅OH) had an identical retention time. After the dialysate probe was implanted in the rat myocardium, the levels of 2,3-DHBA remained unchanged until reperfusion. When the heart was reperfused, a marked elevation of the levels of 2,3-DHBA was observed in the heart dialysate. However, this elevation of 2,3-DHBA was not observed outside the ischemic area. When diltiazem (100 µg ⋅ min21 ⋅ kg21 ) was administered intravenously, the elevation of 2,3-DHBA was not observed in ischemic-reperfused rat heart. Moreover, when allopurinol (10 mg ⋅ kg21 ) was administered by i.v. injection, the elevation of 2,3DHBA was not observed (Fig. 4). DISCUSSION
FIG. 3. Changes in heart rate following systemic administration of diltiazem. The systemic administration of diltiazem gradually decreased heart rate. Values are expressed as mean 6 SE of eight rats. *p , 0.05 or **p , 0.01, significant change. NS, nonsignificant change. Abscissa: administration of diltiazem starting 30 min (120 min after experiment). Oc, occlusion; Rep, reperfusion, s—s: control, n ⋅ ⋅ ⋅ n: diltiazem.
Several experimental studies have shown that oxygen radicals contribute to myocardial damage induced by the ischemia-reperfusion (14,20,22). The occurrence of intracellular Ca21 plays an important role in cardiac dysfunction. The heart subjected to ischemia is attributed to the occurrence of intracellular Ca21 overload (18). Intracellular calcium overload has been reported to be one of the causes of ischemia-reperfusion injury (11). However, the exact mechanisms loading to the development of this abnormality are far from clear. It is well known that diltiazem decreases the blood pres-
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FIG. 4. Effect of diltiazem and allopurinol in ischemic-reper-
fused rat heart by the formation of ⋅OH products of salicylate. In vivo trapping of highly reactive hydroxyl radicals (⋅OH) in extracellular fluid of myocardium was investigated by infusing Ringer’s solution containing microdialysis probe placed in the rat heart. Dialysate samples were collected at 15-min intervals and immediately assayed for 2,3-DHBA using HPLC-EC procedure. Values are expressed as mean 6SE of six rats. *p , 0.01 vs initial level. NS, nonsignificant change. Ische., Ischemia, Rep., reperfusion.
sure and heart rate (19). Diltiazem exhibits different potencies on vascular and cardiac muscles (15). The reduction of the mean arterial blood pressure was achieved by the injection of calcium channel-blocking agents. We applied in vivo microdialysis technique (20) to examine the protective effect of diltiazem on myocardial ischemic injury by preventing ischemic arrhythmia or ⋅OH generation. When the ischemic arrhythmia appeared, the presence of free radicals was observed in the ischemic-reperfused rat heart. However, neither typical changes in ECG nor ⋅OH generation in the ischemic-reperfused rat heart were observed after systemic administration of diltiazem, a calcium antagonist. Free radical formation produced might have contributed to the in vivo free radical formation by Ca21. Ca21 overload may convert xanthine dehydrogenase to xanthine oxidase during ischemia (Fig. 5). Xanthine oxidase resulting from xanthine
FIG. 5. The reaction pathway in rat heart illustrates the for-
mation of hydroxyl radical. Abbreviations: O22. : superoxide anion; ⋅OH: hydroxyl radical; XD: xanthine dehydrogenase; Dilti.: diltiazem; Allo.: allopurinol.
dehydrogenase during ischemia (16), is considered to be a potential source of superoxide in the rat myocardium. Superoxide has an extremely short half-life (7) and rapidly undergoes dismutation yielding H2 O2. H2 O2 then it undergoes a Fenton-type reaction in the presence of iron and yields highly cytotoxic ⋅OH (6). After administration of allopurinol, an xanthine oxidase inhibitor, the incidence of ischemic arrhythmia was not observed. Therefore, with allopurinol administration, ⋅OH generation was not indicated in the ischemic reperfused rat heart (Fig. 4). These results seem to indicate that diltiazem, a calcium antagonist, may suppress the ⋅OH generation from xanthine-xanthine oxidase system by ischemia-reperfusion. In conclusion, we demonstrated the suppression of ⋅OH in the ischemic-reperfused myocardium following systemic administration of diltiazem, a calcium antagonist. Cardiac microdialysis using the hydroxylation of salicylate to detect ⋅OH generation may be useful in answering some of the fundamental questions concerning the relevance of oxidant damage in the pathogenesis of heart disorders. We are thankful to Dr. Hideyuki Takenaga (Tanabe Seiyaku Co., Ltd.) for his valuable discussions. This work was supported by Tanabe Seiyaku Co. Ltd.
References 1. Andreoli, S. Mechanism of endothelial cell ATP depletion after oxidant injury. Pediatr. Res. 25:97–101;1989. 2. Bellamo, G.; Jewell, S.A.; Thor, H.; Orrenius, S. Regulation of intracellular calcium compartmentation. Proc. Natl. Acad. Sci. USA 79:6842–6846;1982. 3. Cao, W.; Carney, J.M.; Duchon, A.; Floyd, R.A.; Chevion, M. Oxygen free radical involvement in ischemia and reperfusion injury to brain. Neurosci. Lett. 88:233–238;1988. 4. Deby, C.; Goutier, R. New perspectives on the biochemistry of superoxide anion and the efficiency of superoxide dismutases. Biochem. Pharmacol. 39:399–405;1990. 5. Floyd, R.A.; Watson, J.J.; Wong, P.K. Sensitive assay of hydroxyl free radical formation utilizing high pressure liquid chromatography with electrochemical detection of phenol and salicylate hydroxylation products. J. Biochem. Biophys. Methods 10:221–235;1984. 6. Gerlach, M.; Ben-Shachar, D.; Riederer, P.; Youdim, M.B.H. Altered brain metabolism of iron as a cause of neurodegenerative disease? J. Neurochem. 63:793–807;1994. 7. Halliwell, B. Oxidants and the central nervous system: some fundamental questions. Is oxidant damage relevant to Parkinson’s disease, Alzheimer’s disease, traumatic injury, or stroke? Acta Neurol. Scand. 126:23–33;1989. 8. Halliwell, B.; Kaur, H.; Ingleman-Sundberg, M. Hydroxylation of salicylate as an assay for hydroxyl radicals: A cautionary note. Free Rad. Biol. Med. 10:439–441;1991. 9. Hearse, D.J.; Humphrey, S.M.; Nayler, W.G.; Slade, A.; Border, D. Ultrastructural damage associated with reoxygenation of the anoxic myocardium. J. Mol. Cell Cardiol. 7:315–324; 1975. 10. Hess, M.L.; Manson, N.H.; Okabe, E. Involvement of free radicals in the pathophysiology of ischemic heart disease. Can. J. Physiol. Pharmacol. 60:1382–1389;1982. 11. Hess, M.L.; Manson, N.H. Molecular oxygen: Friend and foe:
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The role of the oxygen free radical system in the calcium paradox, the oxygen paradox ischemia/reperfusion injury. J. Mol. Cell Cardiol. 16:969–985;1984. Ikeda, Y.; Long, D. M. The molecular basis of brain injury and brain edema: The role of oxygen free radicals. Neurology 27: 1–10;1990. Jolly, S.R.; Kane, W.J.; Baile, M.B.; Abrams, G.D.; Lucchesi, B.R. Canine myocardial reperfusion injury: Its reduction by combined administration of superoxide dismutase and catalase. Cir. Res. 54:277–285;1984. Karmazyn, M. Contribution of prostaglandins to reperfusioninduced ventricular failure in isolated rat heart. Am. J. Physiol. 251:H133–H140;1986. Lathrop, D.A.; Valle-Aguilera, J.R.; Millard, R.W.; Gaum, W.E.; Hannon, D.V.; Francis, P.D.; Nakaya, H.; Schwart, A. Comparative electrophysiologic and coronary hemodynamic effect of diltiazem, nisoldipine and verapamile in myocardial tissue. Am. J. Cardiol. 49:613–620;1982. McCord, J.M. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312:159–163;1985. Minyailenko, T.D.; Pozharrov, V.P.; Serenko, M.M. Severe hypoxia activates lipid peroxidation in the rat brain. Chem. Phys. Lipids. 55:25–28;1990. Murphy, E.; Jacob, R.; Lieberman, M. Cytosolic free calcium in chick cells. J. Mol. Cell Cardiol. 17:221–231;1985. Nagao, T.; Murata, S.; Ikezawa, K.; Iikeo, T.; Narita, H.; Sato, M. Effect of diltiazem on hemodynamics and His bundle elec-
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ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00262-9
Protective Effect of Diltiazem on Myocardial Ischemic Injury Associated With ⋅OH Generation Toshio Obata and Yasumitsu Yamanaka Department of Pharmacology Oita Medical University, Hasama-machi, Oita 879-55, Japan ABSTRACT. We examined the protective effect of diltiazem, a calcium antagonist, on myocardial ischemic injury associated with generation of hydroxyl free radicals (⋅OH). Salicylic acid in Ringer’s solution (0.5 nmol ⋅ µl 21 ⋅ min21 ) was infused directly through a microdialysis probe to detect the generation of ⋅OH as reflected by the formation of 2,3-dihydroxybenzoic acid (DHBA) in the myocardium. Cardiac dialysate was assayed for 2,3-DHBA by a high-performance liquid chromatographic-electrochemical (HPLC-EC) procedure. The heart was subjected to myocardial ischemia for 15 min by occlusion of left anterior descending artery (LAD). The presence of ⋅OH was indicated in the ischemic reperfused rat heart. However, when heart was reperfused, the elevation of 2,3-DHBA by 15-min ischemia was not observed in the ischemic zone following systemic administration of diltiazem (100 µg ⋅ min21 ⋅ kg21 ), a calcium antagonist. When corresponding experiments were performed with allopurinol (10 mg ⋅ kg21 ) administration of i.v. injection, the elevation of 2,3-DHBA was not observed. These results suggest that diltiazem may suppress the ⋅OH generation from xanthine-xanthine oxidase system by ischemia-reperfusion. comp biochem physiol 117A;2:257–261, 1997. 1997 Elsevier Science Inc. KEY WORDS. Allopurinol, calcium overload, diltiazem, hydroxyl free radical, ischemia, microdialysis, salicylic acid
INTRODUCTION The deleterious effects of reperfusion on the ischemic myocardium have been linked to the production of oxygenderived free radicals (9,13,16). Recent studies (10,16,21) suggest an involvement of oxygen free radicals in the pathophysiology of myocardial ischemia–induced cell damage. Intracellular calcium level builds and catalyzes the conversion of xanthine dehydrogenase to xanthine oxidase (26), which produces superoxide. Calcium (Ca21 ) ion may be a very important factor in the induction of irreversible ischemic injury (25). However, the interaction between intracellular Ca21 overload and oxygen free radicals in myocardium is not clear. Intracellular Ca21 level has been proposed as a source of oxidative toxicity (2). Superoxide, either directly or after conversion to hydroxyl radical (4,12), damages biological membrane (17) and other cellular components, including DNA (1), resulting in cell death. The hydroxyl free radical (⋅OH) reacts with salicylate and generates 2,3- and 2,5-dihydroxybenzoic acid (DHBA), which can be measured electrochemically in picomole quantity by high pressure liquid chromatography (HPLC) (5,24). However, 2,3-DHBA can be non-enzymatically Address reprint requests to: Toshio Obata, Department of Pharmacology, Oita Medical University, Hasama-machi, Oita 879-55, Japan. Tel. 097586-5724; Fax 0975-86-5729. Abbreviations—HPLC, high-performance liquid chromatography; 2,3DHBA, 2,3-dihydroxybenzoic acid; ⋅OH, hydroxyl free radical.
formed by the aromatic hydroxylation of ⋅OH and can provide an assay for ⋅OH formation (5,8,24). The present study focused on a possible use of salicylate hydroxylation as an in vivo trapping procedure (3,5,8,23,24) for monitoring the time course of 2,3-DHBA generation in the heart. The present study attempted to examine the protective effect of diltiazem, a calcium antagonist, on myocardial ischemic/reperfusion injury caused by ⋅OH generation. MATERIALS AND METHODS Animals Male Wistar rats weighing 350–450 g were anesthetized with chloral hydrate (400 mg ⋅ kg21 i.p.). The level anesthesia was maintained with continuous intravenous infusion of chloral hydrate (20 mg ⋅ kg21 ⋅ h21 ). Artificial ventilation was maintained with a constant-volume respiration using room air mixed with oxygen. The heart rate, arterial blood pressure, and electrocardiogram (ECG) were monitored and recorded continuously. Heparin sodium (200 U/kg) was administered intravenously before probe implantation; 100 U/ kg was then given every 1 hr to prevent blood coagulation. Diltiazem (Herbesser; Tanabe Seiyaku Co., Ltd., Tokyo, Japan; 100 µg ⋅ min21 ⋅ kg21 ) was continuously administered by intravenous infusion. Changes in heart rate were monitored by minipolygraph (Nihon Kohden, RM-6100, Tokyo, Japan). This study was approved by the Ethical Committee for Animal Experiments, Oita Medical University.
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Cardiac Microdialysis We used an in vivo microdialysis technique (20) to examine the ⋅OH generation on myocardial ischemia/reperfusion injury. With the animal in lateral position, the left fifth or sixth rib on the left side was partially removed to observe the heart. A small incision was made in the pericardium, and, with a fine guiding needle. The dialysis probe was implanted into the ischemic zone of the left anterior descending coronary artery (LAD). The inlet side of the microdialysis probe was connected by a polyethylene tube to a 1-ml syringe, which was driven by a microinjection pump (Carnegie Medicine CMA/100, Sweden). The outlet side of the probe was connected to the same tube and placed into a small collecting tube. Dialysis probe was perfused with Ringer’s solution containing 147 mM NaCl, 2,3 mM CaCl2, 4 mM KCl, pH 7.0. To determine an adequate perfusion speed, we tested in vitro to calculate the recovery of 2,3-DHBA through the membrane under various conditions. The dialysis probe was bathed in 37°C Ringer’s solution with a constant 2,3-DHBA concentration. The dialysate 2,3-DHBA concentration was measured directly by HPLC with electrochemical (EC) procedure. When the probe (3-mm exposure) was perfused with 1 µl ⋅ min21 in vitro, the relative recovery rate of 1 µM 2,3-DHBA was an average of about 10%. Using this perfusion speed in vitro, we obtained uniform relative recovery rates with different 2,3-DHBA concentrations in the testing dialysate solution. On the basis of these results, we chose a perfusion speed of 1 µ21 ⋅ min21 for in vivo experiments. Ringer’s solution containing 0.5 mM salicylic acid (0.5 n mol ⋅ µl21 ⋅ min21 ) was infused directly through the microdialysis probe in the ventricular LAD. The dialysis samples from the myocardium were immediately injected for analysis into an HPLCEC system equipped with a glassy carbon working electrode (EICOM, Kyoto, Japan) and reverse-phase column, an Eicompak MA-50DS column (5 µm, 4,6 3 150 mm, Eicom). The working electrode was set at a detector potential of 0.75 V. Each liter of mobile phase contained 1.5 g heptane sulfonic acid sodium salt (Sigma Chemical, St Louis, MO), 0.1 g Na2 EDTA, 3 ml triethylamine (Wako Pure Chemical Industries, Osaka, Japan) and 125 ml acetonitrile (Wako) dissolved in H2O. The pH of solution was adjusted to 2.8 with 3 ml phosphoric acid (Wako) and filtered through a Millipore filter before the addition of acetonitrile. Flow rate of 0.7 and 0.9 ml ⋅ min21 allowed the sample to be assayed. The dialysate (1 µl ⋅ min21 ) was then collected every 15 min into small collecting tubes containing 15 µl of ice-cold 0.1 N HClO4 and assayed immediately for 2,3DHBA by a HPLC-EC procedure.
tube from the surrounding coronary artery. The heart was subjected to regional ischemia for 15 min by the occlusion of LAD followed by reperfusion. Statistical Methods The results were expressed as the mean SEM of the 2,3DHBA outputs. Statistical analysis were performed by ANOVA test. A P value of ,0.05 was considered significant. RESULTS After a 60-min washout with Ringer’s solution, we determined the time course of norepinephrine levels in a dialysates collected at 15-min intervals over a period of 135 min (Fig. 1). When the dialysate norepinephrine level had reached an almost steady-state level, the heart was subjected to regional ischemia for 15 min by the occlusion of LAD, followed then by reperfusion. It reached a low steady-state level of 0.148 6 0.015 nmol/ml at 150–165 min after probe implantation. When occlusion was begun and reperfusion was started, we confirmed typical changes in ECG in control rats. The incidence of ischemic arrhythmia was observed at 13 sec after occlusion of the coronary artery. Ventricular tachycardia and ventricular fibrillation occurred at 21 sec by reperfusion. Ischemia decreased heart rate and mean arterial pressure. Heart rate decreased from 348 6 25 beats ⋅ min 21 during ischemia to 306 6 27 beats ⋅ min21 after reperfusion, but this change was not significant. Mean arterial pressure significantly decreased from 79 6 14 mmHg during ischemia to 67 6 19 mmHg after reperfusion. After systemic administration of diltiazem (100 µg ⋅ min 21 ⋅ kg21 ) starting
FIG. 1. Time course of dialysate norepinephrine (NE) levels
Preparation of Ischemic Rats After the microdialysis probe implantation in ischemic zone, the LAD branch was clamped by a thread through a
in extracellular fluid of myocardium. Dialysate NE gradually decreased over the first 150 min and then reached low steady levels. Values are expressed as mean 6 SE of six rats. *p , 0.01 significant change.
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FIG. 2. Typical changes in the electrocardiogram (ECG). Left anterior descending (LAD) branch was clamped by thread through a tube from surrounding coronary artery. When the heart was subjected to 15 min of regional ischemia by LAD occlusion, incidence of ischemic and reperfusion arrhythmia were observed (Control). After systemic administration of diltiazem (100 mg ⋅ min21 ⋅ kg21 ), incidence of ischemic arrhythmia was not observed in ischemic reperfused heart (Diltiazem). When corresponding experiments were performed with allopurinol (10 mg ⋅ kg21 ) administration of i.v. injection, the incidence of ischemic arrhythmia was not observed (Allopurinol).
30 min (120 min after experiment), incidence of ischemic arrhythmia was not observed in the ischemic-reperfused heart. When corresponding experiments were performed with allopurinol (10 mg ⋅ kg21) administration of i.v. injection, the incidence of ischemic arrhythmia was not observed (Fig. 2). However, the systemic administration of diltiazem gradually decreased heart rate (Fig. 3). Reperfusion gradually decreased heart rate (from 310 6 28 to 292 6 11 beats ⋅ min21 ) or mean arterial pressure (from 68 6 21 to 65 6 18 mmHg).
Effect of Diltiazem and Allopurinol on ⋅OH Formation in Ischemic-reperfused Rat Heart The presence of ⋅OH was observed in ischemic-reperfused rat heart. Sodium salicylate was infused for 150 min to trap ⋅OH, which was formed by ischemia-reperfusion of the myocardium. The authentic standards of 2,3-DHBA (reaction products of salicylic acid and ⋅OH) had an identical retention time. After the dialysate probe was implanted in the rat myocardium, the levels of 2,3-DHBA remained unchanged until reperfusion. When the heart was reperfused, a marked elevation of the levels of 2,3-DHBA was observed in the heart dialysate. However, this elevation of 2,3-DHBA was not observed outside the ischemic area. When diltiazem (100 µg ⋅ min21 ⋅ kg21 ) was administered intravenously, the elevation of 2,3-DHBA was not observed in ischemic-reperfused rat heart. Moreover, when allopurinol (10 mg ⋅ kg21 ) was administered by i.v. injection, the elevation of 2,3DHBA was not observed (Fig. 4). DISCUSSION
FIG. 3. Changes in heart rate following systemic administration of diltiazem. The systemic administration of diltiazem gradually decreased heart rate. Values are expressed as mean 6 SE of eight rats. *p , 0.05 or **p , 0.01, significant change. NS, nonsignificant change. Abscissa: administration of diltiazem starting 30 min (120 min after experiment). Oc, occlusion; Rep, reperfusion, s—s: control, n ⋅ ⋅ ⋅ n: diltiazem.
Several experimental studies have shown that oxygen radicals contribute to myocardial damage induced by the ischemia-reperfusion (14,20,22). The occurrence of intracellular Ca21 plays an important role in cardiac dysfunction. The heart subjected to ischemia is attributed to the occurrence of intracellular Ca21 overload (18). Intracellular calcium overload has been reported to be one of the causes of ischemia-reperfusion injury (11). However, the exact mechanisms loading to the development of this abnormality are far from clear. It is well known that diltiazem decreases the blood pres-
T. Obata and Y. Yamanaka
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FIG. 4. Effect of diltiazem and allopurinol in ischemic-reper-
fused rat heart by the formation of ⋅OH products of salicylate. In vivo trapping of highly reactive hydroxyl radicals (⋅OH) in extracellular fluid of myocardium was investigated by infusing Ringer’s solution containing microdialysis probe placed in the rat heart. Dialysate samples were collected at 15-min intervals and immediately assayed for 2,3-DHBA using HPLC-EC procedure. Values are expressed as mean 6SE of six rats. *p , 0.01 vs initial level. NS, nonsignificant change. Ische., Ischemia, Rep., reperfusion.
sure and heart rate (19). Diltiazem exhibits different potencies on vascular and cardiac muscles (15). The reduction of the mean arterial blood pressure was achieved by the injection of calcium channel-blocking agents. We applied in vivo microdialysis technique (20) to examine the protective effect of diltiazem on myocardial ischemic injury by preventing ischemic arrhythmia or ⋅OH generation. When the ischemic arrhythmia appeared, the presence of free radicals was observed in the ischemic-reperfused rat heart. However, neither typical changes in ECG nor ⋅OH generation in the ischemic-reperfused rat heart were observed after systemic administration of diltiazem, a calcium antagonist. Free radical formation produced might have contributed to the in vivo free radical formation by Ca21. Ca21 overload may convert xanthine dehydrogenase to xanthine oxidase during ischemia (Fig. 5). Xanthine oxidase resulting from xanthine
FIG. 5. The reaction pathway in rat heart illustrates the for-
mation of hydroxyl radical. Abbreviations: O22. : superoxide anion; ⋅OH: hydroxyl radical; XD: xanthine dehydrogenase; Dilti.: diltiazem; Allo.: allopurinol.
dehydrogenase during ischemia (16), is considered to be a potential source of superoxide in the rat myocardium. Superoxide has an extremely short half-life (7) and rapidly undergoes dismutation yielding H2 O2. H2 O2 then it undergoes a Fenton-type reaction in the presence of iron and yields highly cytotoxic ⋅OH (6). After administration of allopurinol, an xanthine oxidase inhibitor, the incidence of ischemic arrhythmia was not observed. Therefore, with allopurinol administration, ⋅OH generation was not indicated in the ischemic reperfused rat heart (Fig. 4). These results seem to indicate that diltiazem, a calcium antagonist, may suppress the ⋅OH generation from xanthine-xanthine oxidase system by ischemia-reperfusion. In conclusion, we demonstrated the suppression of ⋅OH in the ischemic-reperfused myocardium following systemic administration of diltiazem, a calcium antagonist. Cardiac microdialysis using the hydroxylation of salicylate to detect ⋅OH generation may be useful in answering some of the fundamental questions concerning the relevance of oxidant damage in the pathogenesis of heart disorders. We are thankful to Dr. Hideyuki Takenaga (Tanabe Seiyaku Co., Ltd.) for his valuable discussions. This work was supported by Tanabe Seiyaku Co. Ltd.
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