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H+ exchanger attenuates neutrophil-mediated reperfusion injury
Inhibition of Na+/H + Exchanger Attenuates Neutrophil-Mediated Reperfusion Injury Farid C. Faes, MD, Yoshiki Sawa, MD, Hajime Ichikawa, MD, Yasuhisa Shimazaki, MD, Takeki Ohashi, MD, Hirotsugu Fukuda, MD, Ryota Shirakura, MD, and Hikaru Matsuda, MD First Department of Surgery, Osaka University Medical School, Osaka, Japan
Background. The effect of Na+/H + exchange i n h i b i t i o n in neutrophil-induced reperfusion injury was investigated using a n e w a m i l o r i d e analogue, 5-methyl-Nisobutyl amiloride (MIA). Methods. Rat neutrophils were separated using Percoll gradient. Luminol chemiluminescence intensity of isolated neutrophils was depressed by MIA in a dose-dependent manner. Results. The effect of MIA on n e u t r o p h i l - i n d u c e d reperfusion injury was evaluated in Langendorff-perfused rat hearts subjected to 30 minutes of normothermic ischemia. Postischemic left ventricular developed pressure recovery was depressed by the reperfusion with
neutrophils (60% - 7% to 33% -+ 26%) and was reverted by MIA pretreatment (86% --- 17%, p < 0.05). MIA also improved percent recovery of coronary flow (51% --- 2% to 70% + 13%), reduced creatine kinase (0.28 + 0.1 to 0.085 + 0.03 IU • L -1 • g-~ dry wt), and lactate dehydrogenase leakage (10.6 + 3.8 to 5.16 - 1.3 IU • L -1 • g-1 d r y wt) significantly. The incidence of reperfusion-induced ventricular fibrillation also was reduced by MIA. Conclusions. The i n h i b i t i o n of N a + / H ÷ exchange shows a protective effect against neutrophil-induced reperfusion injury possibly by inhibiting the activation of neutrophils.
is no doubt that activated neutrophils contribute T here to myocardial d a m a g e after reflow by releasing
Material and Methods
deleterious c o m p o n e n t s such as reactive oxygen species (eg, superoxide anion) and other cytotoxic substances a n d b y plugging the coronary capillaries that can affect the m y o c a r d i u m directly [1-4]. Because in both experimental and clinical situations, an activation of neutrophils might counteract the potential benefit of reperfusion, efforts have b e e n m a d e to p r e v e n t this n e u t r o p h i | m e d i a t e d i s c h e m i a - r e p e r f u s i o n injury [2, 5, 6]. The activation of neutrophils is regulated by the intracellular pH (ie, intracellular acidification has been shown to attenuate the activation of leukocytes) [7]. As there is evidence that the Na +/H - exchanger regulates the pH of both platelets a n d neutrophils, we h y p o t h e s i z e d that it is possible to attenuate the leukocyte-induced cellular injury by decreasing intracellular pH using an inhibitor of the Na +/H exchanger. The effects of a new potent amiloride analogue, 5-methyl-N-isobutyl-amiloride (MIA), on neutrophil activation a n d n e u t r o p h i l - m e d i a t e d reperfusion i n j u ~ were tested using isolated rat neutrophils and Langendorff-perfused rat hearts to d e t e r m i n e the role of p H regulation in n e u t r o p h i l - m e d i a t e d reperfusion i n j u ~ .
Accepted for publication March 31, 1995. Address reprint requests to Dr Sawa, First Department of Surgery, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. © 1995 by The Society of Thoracic Surgeons
(Ann Thorac Surg 1995;60:377-81)
Chemiluminescence Measurement Study Neutrophils were isolated from male S p r a g u e - D a w l e y rats (body weight, 300 to 350 g) using a m e t h o d d e s c r i b e d by Yuan and Fleming [8]. Briefly, blood cells were sedim e n t a t e d in H a n k ' s solution containing gelatin (2% /wt). N e u t r o p h i l s w e r e s e p a r a t e d from p o l y m o r p h o n u clear neutrophils by the centrifugation in 70% Percoll. Throughout the procedures, the t e m p e r a t u r e was maintained between 30 ° and 37°C. The addition of plateletpoor plasma (10%, vol/vol) to the solutions m a i n t a i n e d the normal m o r p h o l o g y a n d function of neutrophils. This m e t h o d p r o v i d e d neutrophils of n o r m a l m o r p h o l o g y a n d high yield and purity. Isolated neutrophils (6 to 7 × 106/mL) were incubated in H a n k s ' buffer at 37°C for 120 minutes. C h e m i l u m i n e s c e n c e was m e a s u r e d in four groups (four rats in each group): group 1, control; groups 2, 3, and 4, 0.5, 1.0, and 2.0 m m o l / L of MIA were a d d e d to neutrophils, respectively. A chemiluminescence p h o t o m eter (Luminometer 100; Nition, Chiba, Japan) was used for this m e a s u r e m e n t .
Isolated Rat Heart Study The care of all animals used in these e x p e r i m e n t s complied with the " G u i d e for the Care a n d Use of Laboratory A n i m a l s " p u b l i s h e d by the National Institutes of Health (NIH publication 85-23, revised 1985). Male SpragueDawley rats weighing 300 to 350 g were anesthetized with an intraperitoneal injection of p e n t o b a r b i t a l (50 m g / k g b o d y weight). After intravenous administration of h e p a 0003-4975/95]$9.50 0003-4975(95)00370-Z
378
FAE5 ET AL Na ÷ / H - EXCHANGE AND NEUTROPHIL
rin (1 U/g b o d y weight), hearts were excised and imm e r s e d in perfusion m e d i u m at 4°C. The aorta was c a n n u l a t e d and retrograde arterial perfusion was started at a constant pressure of 100 c m H 2 0 . The heart was h o u s e d in a t e m p e r a t u r e - r e g u l a t e d water-jacketed glass c h a m b e r with perfusion m e d i u m to keep the intramyocardial t e m p e r a t u r e at 37°C. A thin latex balloon connected to the pressure transducer was inserted into the left ventricle t h r o u g h the mitral valve to continuously monitor the left ventricular pressure. End-diastolic pressure was set at 8 to 10 m m Hg by adjusting the balloon volume with water. The intraventricular balloon was kept inflated t h r o u g h o u t the experiment. Krebs-Henseleit bicarbonate buffer containing (in mmol/L) NaC1 118.5; NaHCO3 25.0; KC1 4.8; KH2PO 4 1.2; MgSO 4 1.2; CaC12 2.5; glucose 11.0 was used as a perfusion m e d i u m . The solutions were gassed with 95% 02 and 5% CO 2 to maintain a p H of 7.4. Preischemic ventricular function (left ventricular systolic pressure, heart rate, coronary flow) was m e a s u r e d 15 m i n u t e s after the initiation of the perfusion. The hearts were then subjected to 30 minutes of n o r m o t h e r m i c (37°C) global ischemia followed by a reperfusion for 45 minutes. The coronary effluent was collected during the first 5 minutes of reperfusion to m e a s u r e creatine kinase and lactate d e h y d r o g e n a s e leakage. At the e n d of the reperfusion period, ventricular function and coronary flow were m e a s u r e d again. The postischemic functional recovery was expressed as a percentage of each index to the preischemic value. The e x p e r i m e n t s were d i v i d e d into three groups: group 1, control; group 2, perfusion with neutrophils (1.0 × 106/mL) for the first 5 minutes of reperfusion; group 3, same as group 2 with MIA p r e t r e a t m e n t for 2 minutes before the onset of ischemia. Neutrophils were s e p a r a t e d from the whole blood by Percoll g r a d i e n t as described previously [8]. In groups 2 and 3, the neutrophils susp e n d e d in rat p l a s m a were infused at a rate of I m L / m i n during the first 5 minutes of reperfusion. The total a m o u n t of infused n e u t r o p h i l s / h e a r t was 7 to 8 × 106. Thirty milligrams of MIA was dissolved in 10 mL of D M S O a n d a d d e d to KHBB to make a final concentration 10 mmol/L. This was infused through the side arm attached to the aortic cannula at a rate of 2.5 m L / m i n using a peristaltic p u m p (STC 521; T e r u m o Co, Japan) 2 minutes before ischemia. The concentration of MIA (10 mmol/L) was found to be optimal for functional recovery in our in vitro and in vivo p r e l i m i n a r y experiments.
Data Analysis
Results were expressed as mean _+ s t a n d a r d deviation. For the percentage of s p o n t a n e o u s defibrillation, the X2 test was used. For other parameters, Student's t test was used to c o m p a r e the data between groups 1 a n d 2, and 2 and 3. However, the data b e t w e e n groups 1 and 3, a n d a m o n g the three groups were not c o m p a r e d because of the difference of the degree of myocardial injury in the presence or absence of neutrophils and plasma. Significant differences were defined probabilities for each test of a p value of less than 0.05.
Ann Thorac Surg 1995;60:377-81
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Results
Chemiluminescence Measurement
C h e m i l u m i n e s c e n c e was m e a s u r e d in PMA-activated or p l a s m a - s t i m u l a t e d neutrophils. The chemiluminescence count was d e c r e a s e d b y the application of MIA in a d o s e - d e p e n d e n t m a n n e r suggesting that the inhibition of N a * / H - exchange attenuates the neutrophil activation caused either by PMA a n d plasma. The percentages of the chemiluminescence counts (the chemiluminescence value without MIA as 100%) were a v e r a g e d a n d exp r e s s e d in Figure 1. Isolated R a t H e a r t S t u d y
In group 1, percent recovery of left ventricular d e v e l o p e d pressure was 60% -+ 7% after 30 minutes of n o r m o t h e r mic ischemia. The administration of neutrophils during reperfusion d e t e r i o r a t e d the postischemic contractile function in group 2 (33% _+ 26%). The detrimental effect of neutrophils, however, was inhibited by the administration of MIA in group 3 (86% ÷ 17%, p < 0.05 versus group 2) (Fig 2). The percent recovery of coronary flow in groups 1, 2, a n d 3 were 63% + 23%, 51% + 2%, and 70% *- 13% (p < 0.05 versus group 2), respectively (Fig 3). The activated neutrophils i n d u c e d the elevations of myocardial enzyme leakage (creatine kinase a n d lactate dehydrogenase), which were significantly reverted b y the application of MIA (Figs 4, 5). The application of MIA also reverted the electromechanical a b n o r m a l i t y caused by ischemia reperfusion injury. Ventricular fibrillation on the first 5 m i n u t e s of reperfusion was s p o n t a n e o u s l y reverted to sinus r h y t h m in 3 of 6, 2 of 6, a n d 6 of 6 in groups 1, 2, a n d 3, respectively (Fig 6). G r o u p 3 s h o w e d significantly lower percentage of spontaneous defibrillation than did group 2 (p < 0.05).
Ann Thorac Surg
FAESET AL Na+/H * EXCHANGE AND NEUTROPHIL
1995;60:377-81
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Fig 2. Left ventricular developed pressure (LVDP) after ischemia is expressed as a percentage of preischemic value. The averaged percent recovery is shown. The postischemic percent recovery of the LVDP was significantly higher in group 3 (p < 0.05) than in group 2.
Comment The p r e s e n t study d e m o n s t r a t e d that MIA inhibited the activation of neutrophils in an in vitro e x p e r i m e n t and that preischemic application of MIA attenuated ischerniar e p e r f u s i o n injury in isolated rat hearts r e p e r f u s e d
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Fig 4. Creatine kinase (CK) leakage during reperfusion period. The addition of activated neutrophils in group 2 significantly increased the CK leakage during reperfusion (versus group 1, p < 0.01). The application of MIA in group 3 significantly prevented this neutrophil-induced increase of CK (p < 0.01).
with activated neutrophils after 30 minutes of n o r m o t h e r mic global ischemia. These results suggest that MIA a t t e n u a t e d the postischemic ventricular damage, low reflow, and reperfusion arrhythmias in m y o c a r d i u m by decreasing neutrophil activation. Although the beneficial effects of N a + / H + exchange inhibition on i s c h e m i a - r e p e r f u s i o n injury were described in various experimental m o d e l s in various species [9-12], the exact m e c h a n i s m is still unclear. There are two possible explanations of the present result. First, as r e p o r t e d previously [13, 14], this effect of the inhibition of Na +/H* exchange m a y lead to an inhibition of sodium influx during early reperfusion w h e r e the proton gradient b e t w e e n intraceilular and extracellular space is exac-
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Fig 3. Coronary flow rate after ischemia is expressed as a percentage of preischemic value. The averaged percent coronary flow 45 minutes after ischemia is shown. Coronary flow after 45 minutes of reperfusion was significantly higher in group 3 (p < 0.05) than in group 2.
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Fig 5. Lactate dehydrogenase (LDH) leakage during reperfusion period. The LDH leakage was also significantly prevented by the application of MIA as well as creatine kinase leakage.
380
EAES ET AL Na*/H" EXCHANGEAND NEUTROPHIL
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Ann Thorac Surg 1995;60:377-81
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Fig 6. The incidence of spontaneous dt~fibrillation in the first 5 minutes of reperf~sion. The hearts pretreated with MIA showed spontaneous defibrillation in all cases (100%). In the cases reperfused with neutrophils at &e first moments of reperfilsfim, only two hearts (33%) could reach good contractili~. Group 3 showed sign!ficantly lower percentage of spontaneous d~fibrillatiou than did group 2 (p < 0.05).
erbated. The inhibition of sodium influx would result in the inhibition of C a 2~ influx by way of the Na~/Ca 2 exchange, hence prevented the intracellular C a 2~ o v e r load. These mechanisms are similar to that of the beneficial effects of acidic reperfusion described elsewhere [15, 16]. Theoretically, an inhibition of Na~/Ca 2' exchange can also occur either directly by MIA or by amiloride-induced intracellular acidosis 117, 18]. The MIA used in this study is reported to be 190 times more potent than amiloride and its analogues to inhibit Na + / H exchange a n d has a higher specificity to Na */H* exchange [9-11]. Although the intracellular levels of C a 2~ , Na ~, a n d pH could not be evaluated in this study, it is possible that the preischemic application of MIA attenuated not only the reperfusion-induced i n j u ~ i n d u c e d by neutrophils but also the deterioration during ischemia by decreasing the intracellular Ca 2~ overload [11, 12] in our experiment. The second possible mechanism is the direct inactivation of neutrophils by MIA. The inactivation of neutrophils contributed to this beneficial effect of MIA in our experimental condition as shown by the statistically significant difference between groups 2 and 3, but not groups I and 2. The activation of neutrophils is regulated by the intracellular pH; intracellular acidification has been shown to attenuate the activation of leukocytes [7]. The release of neutrophil products, such as phospholipase A2 and 5-1ipoxygenase, d e p e n d s on the intracellular Ca 2+ [19] and Na ~/H" exchanger is responsible for the elevation of the intracellular C a 2 " . The attenuation of the n e u t r o p h i l - i n d u c e d reperfusion injury in myocardium can be achieved through this inactivation of neu-
trophils by MIA. This is consistent with our in vitro result that the chemiluminescence from neutrophils was decreased by MIA. Therefore, it is possible to attenuate the neutrophil-mediated reperfusion injury by decreasing intracellular pH using an inhibitor of N a ÷ / H ÷ exchanger such as MIA. In the present study, we did not compare the efficacy of MIA between the reperfused heart in the presence a n d absence of neutrophils and plasma to clarify the efficacy of MIA by prevention of intracellular Ca 2+ influx or neutrophil inactivation. There was no significant difference in the recovery between these two groups (data not shown). It appears to be meaningless to compare the data b e t w e e n these two groups because both groups have different levels of myocardial injury in the presence a n d absence of neutrophils and plasma. However, we can speculate from our results and data from other researchers that MIA contributes to the attenuation of reperfusion injury with a combination of both the prevention of Ca influx a n d the inactivation of neutrophil. The m e c h a n i s m of myocardial ischemia reperfusion injury is multifactorial. Therefore, the combination of the myocardial protective intervention appears to attenuate much more the degree of myocardial injury than it did with only one method. Especially, the role of neutrophils in reperfusion injury and the protection against neutrophil-induced injury have attracted much interest [6, 20, 21]. O n the other hand, the m o d u l a t i o n of pH seemed to be a possible candidate to obtain a better myocardial protection in cardiac operations. In the present study, we proved that these two factors act mutually in a neutrophil-reperfused isolated rat heart model. Further investigation is n e e d e d to clarify the exact m e c h a n i s m of reperfusion injury in myocardium and to apply this protective effect of MIA into the clinical situation. This work was supported in part by a grant in aid for scientific research from the Ministry of Education, Science, and Culture of Japan. FCF is a recipient of a scholarship from the Ministry of Education, Science, and Culture of Japan.
References 1. Mullane KM, Westlin W, Kraemer R. Activated neutrophils release mediators that may contribute to myocardial injury and dysfunction associated with ischemia and reperfusion. Ann N Y Acad Sci 1988;524:103-21. 2. Sawa Y, Matsuda H, Shimazaki Y, et al. Evaluation of leukocyte-depleted terminal blood cardioplegic solution in patients undergoing elective and emergency coronary artery bypass grafting. J Thorac Cardiovasc Surg 1994;108 1125-31. 3. Sawa Y, Nakano S, Shimazaki Y, Nishimura M, Kuratani T, Matsuda H. Myocardial protective effect and its mechanism of leukocyte depleted reperfusion in neonatal rabbit hearts. Ann Thorac Surg 1994;58:1386-90. 4. Josephson RA, Silverman HS, Lakatta EG, Stern MD, Zweier JL. Study of the mechanisms of hydrogen peroxide and hydroxyl free radical induced cellular injury and calcium overload in cardiac myocytes. J Biol Chem 1991;266:2354-61. 5. Bednar M, Smith B, Pinto A, Mullane KM. Nafazatrom induced salvage of ischemic myocardium in anesthetized dogs is mediated through inhibition of neutrophil function. Circ Res 1985;57:131-41.
Ann Thorac Surg 1995;60:377-81
6. Sawa Y, Schaper J, Roth M, et al. Platelet-activating factor plays an important role in reperfusion injury in myocardium. Efficacy of platelet-activating factor receptor antagonist (CV-3988) as compared with leukocyte-depleted reperfusion. J Thorac Cardiovasc Surg 1994;108:953-9. 7. Osaki M, Sumimoto H, Takeshige K, Cragoe EJ Jr, Hori Y, Minakami S. Na ÷/H + exchange modulates the production of leukotriene B4 by human neutrophils. Biochem J 1989;257: 751-8. 8. Yuan Y, Fleming BP. A method for isolation and fluorescent labeling of rat neutrophils for intravital microvascular studies. Microvasc Res 1990;40:218-29. 9. Dennis SC, Coetzee WA, Cragoe EJ Jr, Opie LH. Effects of proton buffering and of amiloride derivatives on reperfusion arrhythmias in isolated rat hearts. Possible evidence for an arrhythmogenic role of Nat-H ÷ exchange. Circ Res 1990;66: 1156-9. 10. Talor Z, Ng SC, Cragoe EJ, Arruda JA. Methyl isobutyl amiloride: a new probe to assess the number of Na-H antiporters. Life Sci 1989;45:517-23. 11. Moffat MP, Karmazyn M. Protective effects of the potent Na/H exchange inhibitor methylisobutyl amiloride against post-ischemic contractile dysfunction in rat and guinea-pig hearts. J Mol Cell Cardiol 1993;25:959-71. 12. Karmazyn M. Amiloride enhances postischemic ventricular recovery: possible role of Na*-H- exchange. Am J Physiol 1988;255:H608-15. 13. Lazdunski M, Frelin C, Vigne P. The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal
FAES ET AL Na*/H' EXCHANGEAND NEUTROPHIL
14.
15. 16. 17. 18. 19. 20. 21.
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concentrations of sodium and internal pH. J Mol Cell Cardiol 1985;17:1029-41. Tani M, Neely JR. Role of intracellular Na * in Ca2÷ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H+-Na ÷ and Na+-Ca 2- exchange. Circ Res 1989;65:1045-56. Avkiran M, Ibuki C. Reperfusion-induced arrhythmias. A role for washout of extracellular protons? Circ Res 1992;71: 1429-40. Kitakaze M, Weisfeldt ML, Marban E. Acidosis during early reperfusion prevents myocardial stunning in perfused ferret hearts. J Clin Invest 1988;82:920-7. Earm YE, Irisawa H. Effects of pH on the Na-Ca exchange current in single ventricular cells of the guinea pig. Jpn Heart J 1986;27(Suppl 1):153-8. Doering AE, Lederer WJ. The mechanism by which cytoplasmic protons inhibit the sodium-calcium exchanger in guinea-pig heart cells. J Physiol 1993;466:481-99. Tripp CS, Mahoney M, Needleman P. Calcium ionophore enables soluble agonists to stimulate macrophage 5-1ipoxygenase. J Biol Chem 1985;260:5895-8. Wachtfogel YT, Kucich U, Greenp|ate J, et al. Human neutrophil degranulation during extracorporeal circulation. Blood 1987;69:324-30. Byrne JG, Smith wJ, Murphy MP, Couper GS, Appleyard RF, Cohn LH. Complete prevention of myocardial stunning, contracture, low-reflow, and edema after heart transplantation by blocking neutrophil adhesion molecules during reperfusion. J Thorac Cardiovasc Surg 1992;104:1589-96.
Notice From the American Board of Thoracic Surgery The part I (written) examination will be held at the Atlanta Airport Hilton and Towers, Atlanta Airport, Atlanta, GA, on February 9, 1997. The closing date for registration is August 1, 1996. To be admissible for the part II (oral) examination, a candidate must have successfully c o m p l e t e d the part I (written) examination.
A candidate applying for admission to the certifying examination must fulfill all the r e q u i r e m e n t s of the board in force at the time the application is received. Please address all communications to the A m e r i c a n Board of Thoracic Surgery, O n e Rotary Center, Suite 803, Evanston, IL 60201.
Background. The effect of Na+/H + exchange i n h i b i t i o n in neutrophil-induced reperfusion injury was investigated using a n e w a m i l o r i d e analogue, 5-methyl-Nisobutyl amiloride (MIA). Methods. Rat neutrophils were separated using Percoll gradient. Luminol chemiluminescence intensity of isolated neutrophils was depressed by MIA in a dose-dependent manner. Results. The effect of MIA on n e u t r o p h i l - i n d u c e d reperfusion injury was evaluated in Langendorff-perfused rat hearts subjected to 30 minutes of normothermic ischemia. Postischemic left ventricular developed pressure recovery was depressed by the reperfusion with
neutrophils (60% - 7% to 33% -+ 26%) and was reverted by MIA pretreatment (86% --- 17%, p < 0.05). MIA also improved percent recovery of coronary flow (51% --- 2% to 70% + 13%), reduced creatine kinase (0.28 + 0.1 to 0.085 + 0.03 IU • L -1 • g-~ dry wt), and lactate dehydrogenase leakage (10.6 + 3.8 to 5.16 - 1.3 IU • L -1 • g-1 d r y wt) significantly. The incidence of reperfusion-induced ventricular fibrillation also was reduced by MIA. Conclusions. The i n h i b i t i o n of N a + / H ÷ exchange shows a protective effect against neutrophil-induced reperfusion injury possibly by inhibiting the activation of neutrophils.
is no doubt that activated neutrophils contribute T here to myocardial d a m a g e after reflow by releasing
Material and Methods
deleterious c o m p o n e n t s such as reactive oxygen species (eg, superoxide anion) and other cytotoxic substances a n d b y plugging the coronary capillaries that can affect the m y o c a r d i u m directly [1-4]. Because in both experimental and clinical situations, an activation of neutrophils might counteract the potential benefit of reperfusion, efforts have b e e n m a d e to p r e v e n t this n e u t r o p h i | m e d i a t e d i s c h e m i a - r e p e r f u s i o n injury [2, 5, 6]. The activation of neutrophils is regulated by the intracellular pH (ie, intracellular acidification has been shown to attenuate the activation of leukocytes) [7]. As there is evidence that the Na +/H - exchanger regulates the pH of both platelets a n d neutrophils, we h y p o t h e s i z e d that it is possible to attenuate the leukocyte-induced cellular injury by decreasing intracellular pH using an inhibitor of the Na +/H exchanger. The effects of a new potent amiloride analogue, 5-methyl-N-isobutyl-amiloride (MIA), on neutrophil activation a n d n e u t r o p h i l - m e d i a t e d reperfusion i n j u ~ were tested using isolated rat neutrophils and Langendorff-perfused rat hearts to d e t e r m i n e the role of p H regulation in n e u t r o p h i l - m e d i a t e d reperfusion i n j u ~ .
Accepted for publication March 31, 1995. Address reprint requests to Dr Sawa, First Department of Surgery, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. © 1995 by The Society of Thoracic Surgeons
(Ann Thorac Surg 1995;60:377-81)
Chemiluminescence Measurement Study Neutrophils were isolated from male S p r a g u e - D a w l e y rats (body weight, 300 to 350 g) using a m e t h o d d e s c r i b e d by Yuan and Fleming [8]. Briefly, blood cells were sedim e n t a t e d in H a n k ' s solution containing gelatin (2% /wt). N e u t r o p h i l s w e r e s e p a r a t e d from p o l y m o r p h o n u clear neutrophils by the centrifugation in 70% Percoll. Throughout the procedures, the t e m p e r a t u r e was maintained between 30 ° and 37°C. The addition of plateletpoor plasma (10%, vol/vol) to the solutions m a i n t a i n e d the normal m o r p h o l o g y a n d function of neutrophils. This m e t h o d p r o v i d e d neutrophils of n o r m a l m o r p h o l o g y a n d high yield and purity. Isolated neutrophils (6 to 7 × 106/mL) were incubated in H a n k s ' buffer at 37°C for 120 minutes. C h e m i l u m i n e s c e n c e was m e a s u r e d in four groups (four rats in each group): group 1, control; groups 2, 3, and 4, 0.5, 1.0, and 2.0 m m o l / L of MIA were a d d e d to neutrophils, respectively. A chemiluminescence p h o t o m eter (Luminometer 100; Nition, Chiba, Japan) was used for this m e a s u r e m e n t .
Isolated Rat Heart Study The care of all animals used in these e x p e r i m e n t s complied with the " G u i d e for the Care a n d Use of Laboratory A n i m a l s " p u b l i s h e d by the National Institutes of Health (NIH publication 85-23, revised 1985). Male SpragueDawley rats weighing 300 to 350 g were anesthetized with an intraperitoneal injection of p e n t o b a r b i t a l (50 m g / k g b o d y weight). After intravenous administration of h e p a 0003-4975/95]$9.50 0003-4975(95)00370-Z
378
FAE5 ET AL Na ÷ / H - EXCHANGE AND NEUTROPHIL
rin (1 U/g b o d y weight), hearts were excised and imm e r s e d in perfusion m e d i u m at 4°C. The aorta was c a n n u l a t e d and retrograde arterial perfusion was started at a constant pressure of 100 c m H 2 0 . The heart was h o u s e d in a t e m p e r a t u r e - r e g u l a t e d water-jacketed glass c h a m b e r with perfusion m e d i u m to keep the intramyocardial t e m p e r a t u r e at 37°C. A thin latex balloon connected to the pressure transducer was inserted into the left ventricle t h r o u g h the mitral valve to continuously monitor the left ventricular pressure. End-diastolic pressure was set at 8 to 10 m m Hg by adjusting the balloon volume with water. The intraventricular balloon was kept inflated t h r o u g h o u t the experiment. Krebs-Henseleit bicarbonate buffer containing (in mmol/L) NaC1 118.5; NaHCO3 25.0; KC1 4.8; KH2PO 4 1.2; MgSO 4 1.2; CaC12 2.5; glucose 11.0 was used as a perfusion m e d i u m . The solutions were gassed with 95% 02 and 5% CO 2 to maintain a p H of 7.4. Preischemic ventricular function (left ventricular systolic pressure, heart rate, coronary flow) was m e a s u r e d 15 m i n u t e s after the initiation of the perfusion. The hearts were then subjected to 30 minutes of n o r m o t h e r m i c (37°C) global ischemia followed by a reperfusion for 45 minutes. The coronary effluent was collected during the first 5 minutes of reperfusion to m e a s u r e creatine kinase and lactate d e h y d r o g e n a s e leakage. At the e n d of the reperfusion period, ventricular function and coronary flow were m e a s u r e d again. The postischemic functional recovery was expressed as a percentage of each index to the preischemic value. The e x p e r i m e n t s were d i v i d e d into three groups: group 1, control; group 2, perfusion with neutrophils (1.0 × 106/mL) for the first 5 minutes of reperfusion; group 3, same as group 2 with MIA p r e t r e a t m e n t for 2 minutes before the onset of ischemia. Neutrophils were s e p a r a t e d from the whole blood by Percoll g r a d i e n t as described previously [8]. In groups 2 and 3, the neutrophils susp e n d e d in rat p l a s m a were infused at a rate of I m L / m i n during the first 5 minutes of reperfusion. The total a m o u n t of infused n e u t r o p h i l s / h e a r t was 7 to 8 × 106. Thirty milligrams of MIA was dissolved in 10 mL of D M S O a n d a d d e d to KHBB to make a final concentration 10 mmol/L. This was infused through the side arm attached to the aortic cannula at a rate of 2.5 m L / m i n using a peristaltic p u m p (STC 521; T e r u m o Co, Japan) 2 minutes before ischemia. The concentration of MIA (10 mmol/L) was found to be optimal for functional recovery in our in vitro and in vivo p r e l i m i n a r y experiments.
Data Analysis
Results were expressed as mean _+ s t a n d a r d deviation. For the percentage of s p o n t a n e o u s defibrillation, the X2 test was used. For other parameters, Student's t test was used to c o m p a r e the data between groups 1 a n d 2, and 2 and 3. However, the data b e t w e e n groups 1 and 3, a n d a m o n g the three groups were not c o m p a r e d because of the difference of the degree of myocardial injury in the presence or absence of neutrophils and plasma. Significant differences were defined probabilities for each test of a p value of less than 0.05.
Ann Thorac Surg 1995;60:377-81
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Results
Chemiluminescence Measurement
C h e m i l u m i n e s c e n c e was m e a s u r e d in PMA-activated or p l a s m a - s t i m u l a t e d neutrophils. The chemiluminescence count was d e c r e a s e d b y the application of MIA in a d o s e - d e p e n d e n t m a n n e r suggesting that the inhibition of N a * / H - exchange attenuates the neutrophil activation caused either by PMA a n d plasma. The percentages of the chemiluminescence counts (the chemiluminescence value without MIA as 100%) were a v e r a g e d a n d exp r e s s e d in Figure 1. Isolated R a t H e a r t S t u d y
In group 1, percent recovery of left ventricular d e v e l o p e d pressure was 60% -+ 7% after 30 minutes of n o r m o t h e r mic ischemia. The administration of neutrophils during reperfusion d e t e r i o r a t e d the postischemic contractile function in group 2 (33% _+ 26%). The detrimental effect of neutrophils, however, was inhibited by the administration of MIA in group 3 (86% ÷ 17%, p < 0.05 versus group 2) (Fig 2). The percent recovery of coronary flow in groups 1, 2, a n d 3 were 63% + 23%, 51% + 2%, and 70% *- 13% (p < 0.05 versus group 2), respectively (Fig 3). The activated neutrophils i n d u c e d the elevations of myocardial enzyme leakage (creatine kinase a n d lactate dehydrogenase), which were significantly reverted b y the application of MIA (Figs 4, 5). The application of MIA also reverted the electromechanical a b n o r m a l i t y caused by ischemia reperfusion injury. Ventricular fibrillation on the first 5 m i n u t e s of reperfusion was s p o n t a n e o u s l y reverted to sinus r h y t h m in 3 of 6, 2 of 6, a n d 6 of 6 in groups 1, 2, a n d 3, respectively (Fig 6). G r o u p 3 s h o w e d significantly lower percentage of spontaneous defibrillation than did group 2 (p < 0.05).
Ann Thorac Surg
FAESET AL Na+/H * EXCHANGE AND NEUTROPHIL
1995;60:377-81
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Fig 2. Left ventricular developed pressure (LVDP) after ischemia is expressed as a percentage of preischemic value. The averaged percent recovery is shown. The postischemic percent recovery of the LVDP was significantly higher in group 3 (p < 0.05) than in group 2.
Comment The p r e s e n t study d e m o n s t r a t e d that MIA inhibited the activation of neutrophils in an in vitro e x p e r i m e n t and that preischemic application of MIA attenuated ischerniar e p e r f u s i o n injury in isolated rat hearts r e p e r f u s e d
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Fig 4. Creatine kinase (CK) leakage during reperfusion period. The addition of activated neutrophils in group 2 significantly increased the CK leakage during reperfusion (versus group 1, p < 0.01). The application of MIA in group 3 significantly prevented this neutrophil-induced increase of CK (p < 0.01).
with activated neutrophils after 30 minutes of n o r m o t h e r mic global ischemia. These results suggest that MIA a t t e n u a t e d the postischemic ventricular damage, low reflow, and reperfusion arrhythmias in m y o c a r d i u m by decreasing neutrophil activation. Although the beneficial effects of N a + / H + exchange inhibition on i s c h e m i a - r e p e r f u s i o n injury were described in various experimental m o d e l s in various species [9-12], the exact m e c h a n i s m is still unclear. There are two possible explanations of the present result. First, as r e p o r t e d previously [13, 14], this effect of the inhibition of Na +/H* exchange m a y lead to an inhibition of sodium influx during early reperfusion w h e r e the proton gradient b e t w e e n intraceilular and extracellular space is exac-
r P< 0.05 7
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Fig 3. Coronary flow rate after ischemia is expressed as a percentage of preischemic value. The averaged percent coronary flow 45 minutes after ischemia is shown. Coronary flow after 45 minutes of reperfusion was significantly higher in group 3 (p < 0.05) than in group 2.
O-
Group-1
Group-2
Group-3
Fig 5. Lactate dehydrogenase (LDH) leakage during reperfusion period. The LDH leakage was also significantly prevented by the application of MIA as well as creatine kinase leakage.
380
EAES ET AL Na*/H" EXCHANGEAND NEUTROPHIL
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Ann Thorac Surg 1995;60:377-81
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Fig 6. The incidence of spontaneous dt~fibrillation in the first 5 minutes of reperf~sion. The hearts pretreated with MIA showed spontaneous defibrillation in all cases (100%). In the cases reperfused with neutrophils at &e first moments of reperfilsfim, only two hearts (33%) could reach good contractili~. Group 3 showed sign!ficantly lower percentage of spontaneous d~fibrillatiou than did group 2 (p < 0.05).
erbated. The inhibition of sodium influx would result in the inhibition of C a 2~ influx by way of the Na~/Ca 2 exchange, hence prevented the intracellular C a 2~ o v e r load. These mechanisms are similar to that of the beneficial effects of acidic reperfusion described elsewhere [15, 16]. Theoretically, an inhibition of Na~/Ca 2' exchange can also occur either directly by MIA or by amiloride-induced intracellular acidosis 117, 18]. The MIA used in this study is reported to be 190 times more potent than amiloride and its analogues to inhibit Na + / H exchange a n d has a higher specificity to Na */H* exchange [9-11]. Although the intracellular levels of C a 2~ , Na ~, a n d pH could not be evaluated in this study, it is possible that the preischemic application of MIA attenuated not only the reperfusion-induced i n j u ~ i n d u c e d by neutrophils but also the deterioration during ischemia by decreasing the intracellular Ca 2~ overload [11, 12] in our experiment. The second possible mechanism is the direct inactivation of neutrophils by MIA. The inactivation of neutrophils contributed to this beneficial effect of MIA in our experimental condition as shown by the statistically significant difference between groups 2 and 3, but not groups I and 2. The activation of neutrophils is regulated by the intracellular pH; intracellular acidification has been shown to attenuate the activation of leukocytes [7]. The release of neutrophil products, such as phospholipase A2 and 5-1ipoxygenase, d e p e n d s on the intracellular Ca 2+ [19] and Na ~/H" exchanger is responsible for the elevation of the intracellular C a 2 " . The attenuation of the n e u t r o p h i l - i n d u c e d reperfusion injury in myocardium can be achieved through this inactivation of neu-
trophils by MIA. This is consistent with our in vitro result that the chemiluminescence from neutrophils was decreased by MIA. Therefore, it is possible to attenuate the neutrophil-mediated reperfusion injury by decreasing intracellular pH using an inhibitor of N a ÷ / H ÷ exchanger such as MIA. In the present study, we did not compare the efficacy of MIA between the reperfused heart in the presence a n d absence of neutrophils and plasma to clarify the efficacy of MIA by prevention of intracellular Ca 2+ influx or neutrophil inactivation. There was no significant difference in the recovery between these two groups (data not shown). It appears to be meaningless to compare the data b e t w e e n these two groups because both groups have different levels of myocardial injury in the presence a n d absence of neutrophils and plasma. However, we can speculate from our results and data from other researchers that MIA contributes to the attenuation of reperfusion injury with a combination of both the prevention of Ca influx a n d the inactivation of neutrophil. The m e c h a n i s m of myocardial ischemia reperfusion injury is multifactorial. Therefore, the combination of the myocardial protective intervention appears to attenuate much more the degree of myocardial injury than it did with only one method. Especially, the role of neutrophils in reperfusion injury and the protection against neutrophil-induced injury have attracted much interest [6, 20, 21]. O n the other hand, the m o d u l a t i o n of pH seemed to be a possible candidate to obtain a better myocardial protection in cardiac operations. In the present study, we proved that these two factors act mutually in a neutrophil-reperfused isolated rat heart model. Further investigation is n e e d e d to clarify the exact m e c h a n i s m of reperfusion injury in myocardium and to apply this protective effect of MIA into the clinical situation. This work was supported in part by a grant in aid for scientific research from the Ministry of Education, Science, and Culture of Japan. FCF is a recipient of a scholarship from the Ministry of Education, Science, and Culture of Japan.
References 1. Mullane KM, Westlin W, Kraemer R. Activated neutrophils release mediators that may contribute to myocardial injury and dysfunction associated with ischemia and reperfusion. Ann N Y Acad Sci 1988;524:103-21. 2. Sawa Y, Matsuda H, Shimazaki Y, et al. Evaluation of leukocyte-depleted terminal blood cardioplegic solution in patients undergoing elective and emergency coronary artery bypass grafting. J Thorac Cardiovasc Surg 1994;108 1125-31. 3. Sawa Y, Nakano S, Shimazaki Y, Nishimura M, Kuratani T, Matsuda H. Myocardial protective effect and its mechanism of leukocyte depleted reperfusion in neonatal rabbit hearts. Ann Thorac Surg 1994;58:1386-90. 4. Josephson RA, Silverman HS, Lakatta EG, Stern MD, Zweier JL. Study of the mechanisms of hydrogen peroxide and hydroxyl free radical induced cellular injury and calcium overload in cardiac myocytes. J Biol Chem 1991;266:2354-61. 5. Bednar M, Smith B, Pinto A, Mullane KM. Nafazatrom induced salvage of ischemic myocardium in anesthetized dogs is mediated through inhibition of neutrophil function. Circ Res 1985;57:131-41.
Ann Thorac Surg 1995;60:377-81
6. Sawa Y, Schaper J, Roth M, et al. Platelet-activating factor plays an important role in reperfusion injury in myocardium. Efficacy of platelet-activating factor receptor antagonist (CV-3988) as compared with leukocyte-depleted reperfusion. J Thorac Cardiovasc Surg 1994;108:953-9. 7. Osaki M, Sumimoto H, Takeshige K, Cragoe EJ Jr, Hori Y, Minakami S. Na ÷/H + exchange modulates the production of leukotriene B4 by human neutrophils. Biochem J 1989;257: 751-8. 8. Yuan Y, Fleming BP. A method for isolation and fluorescent labeling of rat neutrophils for intravital microvascular studies. Microvasc Res 1990;40:218-29. 9. Dennis SC, Coetzee WA, Cragoe EJ Jr, Opie LH. Effects of proton buffering and of amiloride derivatives on reperfusion arrhythmias in isolated rat hearts. Possible evidence for an arrhythmogenic role of Nat-H ÷ exchange. Circ Res 1990;66: 1156-9. 10. Talor Z, Ng SC, Cragoe EJ, Arruda JA. Methyl isobutyl amiloride: a new probe to assess the number of Na-H antiporters. Life Sci 1989;45:517-23. 11. Moffat MP, Karmazyn M. Protective effects of the potent Na/H exchange inhibitor methylisobutyl amiloride against post-ischemic contractile dysfunction in rat and guinea-pig hearts. J Mol Cell Cardiol 1993;25:959-71. 12. Karmazyn M. Amiloride enhances postischemic ventricular recovery: possible role of Na*-H- exchange. Am J Physiol 1988;255:H608-15. 13. Lazdunski M, Frelin C, Vigne P. The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal
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14.
15. 16. 17. 18. 19. 20. 21.
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concentrations of sodium and internal pH. J Mol Cell Cardiol 1985;17:1029-41. Tani M, Neely JR. Role of intracellular Na * in Ca2÷ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H+-Na ÷ and Na+-Ca 2- exchange. Circ Res 1989;65:1045-56. Avkiran M, Ibuki C. Reperfusion-induced arrhythmias. A role for washout of extracellular protons? Circ Res 1992;71: 1429-40. Kitakaze M, Weisfeldt ML, Marban E. Acidosis during early reperfusion prevents myocardial stunning in perfused ferret hearts. J Clin Invest 1988;82:920-7. Earm YE, Irisawa H. Effects of pH on the Na-Ca exchange current in single ventricular cells of the guinea pig. Jpn Heart J 1986;27(Suppl 1):153-8. Doering AE, Lederer WJ. The mechanism by which cytoplasmic protons inhibit the sodium-calcium exchanger in guinea-pig heart cells. J Physiol 1993;466:481-99. Tripp CS, Mahoney M, Needleman P. Calcium ionophore enables soluble agonists to stimulate macrophage 5-1ipoxygenase. J Biol Chem 1985;260:5895-8. Wachtfogel YT, Kucich U, Greenp|ate J, et al. Human neutrophil degranulation during extracorporeal circulation. Blood 1987;69:324-30. Byrne JG, Smith wJ, Murphy MP, Couper GS, Appleyard RF, Cohn LH. Complete prevention of myocardial stunning, contracture, low-reflow, and edema after heart transplantation by blocking neutrophil adhesion molecules during reperfusion. J Thorac Cardiovasc Surg 1992;104:1589-96.
Notice From the American Board of Thoracic Surgery The part I (written) examination will be held at the Atlanta Airport Hilton and Towers, Atlanta Airport, Atlanta, GA, on February 9, 1997. The closing date for registration is August 1, 1996. To be admissible for the part II (oral) examination, a candidate must have successfully c o m p l e t e d the part I (written) examination.
A candidate applying for admission to the certifying examination must fulfill all the r e q u i r e m e n t s of the board in force at the time the application is received. Please address all communications to the A m e r i c a n Board of Thoracic Surgery, O n e Rotary Center, Suite 803, Evanston, IL 60201.