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Ann Thorac Surg 1995;60:377-381
© 1995 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

Inhibition of Na⁺/H⁺ Exchanger Attenuates Neutrophil-Mediated Reperfusion Injury

First Department of Surgery, Osaka University Medical School, Osaka, Japan

Accepted for publication March 31, 1995.

	Abstract

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Background. The effect of Na⁺/H⁺ exchange inhibition in neutrophil-induced reperfusion injury was investigated using a new amiloride analogue, 5-methyl-N-isobutyl 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 neutrophil-induced 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^-1 dry wt), and lactate dehydrogenase leakage (10.6 ± 3.8 to 5.16 ± 1.3 IU • L^-1 • g^-1 dry wt) significantly. The incidence of reperfusion-induced ventricular fibrillation also was reduced by MIA.

Conclusions. The inhibition of Na⁺/H⁺ exchange shows a protective effect against neutrophil-induced reperfusion injury possibly by inhibiting the activation of neutrophils.

	Introduction

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
There is no doubt that activated neutrophils contribute to myocardial damage after reflow by releasing deleterious components such as reactive oxygen species (eg, superoxide anion) and other cytotoxic substances and by plugging the coronary capillaries that can affect the myocardium directly [1–4]. Because in both experimental and clinical situations, an activation of neutrophils might counteract the potential benefit of reperfusion, efforts have been made to prevent this neutrophil-mediated ischemia–reperfusion 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 and neutrophils, we hypothesized 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 and neutrophil-mediated reperfusion injury were tested using isolated rat neutrophils and Langendorff-perfused rat hearts to determine the role of pH regulation in neutrophil-mediated reperfusion injury.

	Material and Methods

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Chemiluminescence Measurement Study
Neutrophils were isolated from male Sprague-Dawley rats (body weight, 300 to 350 g) using a method described by Yuan and Fleming [8]. Briefly, blood cells were sedimentated in Hank's solution containing gelatin (2% /wt). Neutrophils were separated from polymorphonuclear neutrophils by the centrifugation in 70% Percoll. Throughout the procedures, the temperature was maintained between 30° and 37°C. The addition of platelet-poor plasma (10%, vol/vol) to the solutions maintained the normal morphology and function of neutrophils. This method provided neutrophils of normal morphology and high yield and purity. Isolated neutrophils (6 to 7 x 10⁶/mL) were incubated in Hanks' buffer at 37°C for 120 minutes. Chemiluminescence was measured in four groups (four rats in each group): group 1, control; groups 2, 3, and 4, 0.5, 1.0, and 2.0 mmol/L of MIA were added to neutrophils, respectively. A chemiluminescence photometer (Luminometer 100; Nition, Chiba, Japan) was used for this measurement.

Isolated Rat Heart Study
The care of all animals used in these experiments complied with the ``Guide for the Care and Use of Laboratory Animals'' published by the National Institutes of Health (NIH publication 85-23, revised 1985). Male Sprague-Dawley rats weighing 300 to 350 g were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg body weight). After intravenous administration of heparin (1 U/g body weight), hearts were excised and immersed in perfusion medium at 4°C. The aorta was cannulated and retrograde arterial perfusion was started at a constant pressure of 100 cm H₂O. The heart was housed in a temperature-regulated water-jacketed glass chamber with perfusion medium to keep the intramyocardial temperature at 37°C. A thin latex balloon connected to the pressure transducer was inserted into the left ventricle through the mitral valve to continuously monitor the left ventricular pressure. End-diastolic pressure was set at 8 to 10 mm Hg by adjusting the balloon volume with water. The intraventricular balloon was kept inflated throughout the experiment. Krebs-Henseleit bicarbonate buffer containing (in mmol/L) NaCl 118.5; NaHCO₃ 25.0; KCl 4.8; KH₂PO₄ 1.2; MgSO₄ 1.2; CaCl₂ 2.5; glucose 11.0 was used as a perfusion medium. The solutions were gassed with 95% O₂ and 5% CO₂ to maintain a pH of 7.4. Preischemic ventricular function (left ventricular systolic pressure, heart rate, coronary flow) was measured 15 minutes after the initiation of the perfusion. The hearts were then subjected to 30 minutes of normothermic (37°C) global ischemia followed by a reperfusion for 45 minutes. The coronary effluent was collected during the first 5 minutes of reperfusion to measure creatine kinase and lactate dehydrogenase leakage. At the end of the reperfusion period, ventricular function and coronary flow were measured again. The postischemic functional recovery was expressed as a percentage of each index to the preischemic value.

The experiments were divided into three groups: group 1, control; group 2, perfusion with neutrophils (1.0 x 10⁶/mL) for the first 5 minutes of reperfusion; group 3, same as group 2 with MIA pretreatment for 2 minutes before the onset of ischemia. Neutrophils were separated from the whole blood by Percoll gradient as described previously [8]. In groups 2 and 3, the neutrophils suspended in rat plasma were infused at a rate of 1 mL/min during the first 5 minutes of reperfusion. The total amount of infused neutrophils/heart was 7 to 8 x 10⁶.

Thirty milligrams of MIA was dissolved in 10 mL of DMSO and added 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 mL/min using a peristaltic pump (STC 521; Terumo 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 preliminary experiments.

Data Analysis
Results were expressed as mean ± standard deviation. For the percentage of spontaneous defibrillation, the ² test was used. For other parameters, Student's t test was used to compare the data between groups 1 and 2, and 2 and 3. However, the data between groups 1 and 3, and among the three groups were not compared 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.

	Results

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Chemiluminescence Measurement
Chemiluminescence was measured in PMA-activated or plasma-stimulated neutrophils. The chemiluminescence count was decreased by the application of MIA in a dose-dependent manner suggesting that the inhibition of Na⁺/H⁺ exchange attenuates the neutrophil activation caused either by PMA and plasma. The percentages of the chemiluminescence counts (the chemiluminescence value without MIA as 100%) were averaged and expressed in Figure 1.

View larger version (29K): [in this window] [in a new window]   Fig 1. . Chemiluminescence intensity of the neutrophils activated by either the PMA or plasma was decreased by the application of MIA with a dose-dependent manner. Changes in the chemiluminescence intensity were shown as a percent.

View larger version (21K): [in this window] [in a new window]   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.

View larger version (19K): [in this window] [in a new window]   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.

View larger version (19K): [in this window] [in a new window]   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).

View larger version (18K): [in this window] [in a new window]   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.

View larger version (20K): [in this window] [in a new window]   Fig 6. . The incidence of spontaneous defibrillation in the first 5 minutes of reperfusion. The hearts pretreated with MIA showed spontaneous defibrillation in all cases (100%). In the cases reperfused with neutrophils at the first moments of reperfusion, only two hearts (33%) could reach good contractility. Group 3 showed significantly lower percentage of spontaneous defibrillation than did group 2 (p < 0.05).

	Comment

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

Although the beneficial effects of Na⁺/H⁺ exchange inhibition on ischemia–reperfusion injury were described in various experimental models in various species [9–12], the exact mechanism is still unclear. There are two possible explanations of the present result. First, as reported previously [13, 14], this effect of the inhibition of Na⁺/H⁺ exchange may lead to an inhibition of sodium influx during early reperfusion where the proton gradient between intracellular and extracellular space is exacerbated. The inhibition of sodium influx would result in the inhibition of Ca²⁺ influx by way of the Na⁺/Ca²⁺ exchange, hence prevented the intracellular Ca²⁺ overload. These mechanisms are similar to that of the beneficial effects of acidic reperfusion described elsewhere [15, 16]. Theoretically, an inhibition of Na⁺/Ca²⁺ exchange can also occur either directly by MIA or by amiloride-induced intracellular acidosis [17, 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 and has a higher specificity to Na⁺/H⁺ exchange [9–11]. Although the intracellular levels of Ca²⁺, Na⁺, and pH could not be evaluated in this study, it is possible that the preischemic application of MIA attenuated not only the reperfusion-induced injury induced by neutrophils but also the deterioration during ischemia by decreasing the intracellular Ca²⁺ 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 1 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-lipoxygenase, depends on the intracellular Ca²⁺ [19] and Na⁺/H⁺ exchanger is responsible for the elevation of the intracellular Ca²⁺. The attenuation of the neutrophil-induced reperfusion injury in myocardium can be achieved through this inactivation of neutrophils 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 Na⁺/H⁺ exchanger such as MIA.

In the present study, we did not compare the efficacy of MIA between the reperfused heart in the presence and absence of neutrophils and plasma to clarify the efficacy of MIA by prevention of intracellular Ca²⁺ 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 between these two groups because both groups have different levels of myocardial injury in the presence and 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 and the inactivation of neutrophil.

The mechanism 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]. On the other hand, the modulation 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 needed to clarify the exact mechanism of reperfusion injury in myocardium and to apply this protective effect of MIA into the clinical situation.

	Acknowledgments

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
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.

	Footnotes

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 
Address reprint requests to Dr Sawa, First Department of Surgery, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan.

	References

Top Footnotes Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Yoshiki Sawa Hajime Ichikawa Yasuhisa Shimazaki Takeki Ohashi Hirotsugu Fukuda Ryota Shirakura Hikaru Matsuda Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Faes, F. C. Articles by Matsuda, H. Search for Related Content PubMed PubMed Citation Articles by Faes, F. C. Articles by Matsuda, H.