© 2002 American Thoracic Society
Effect of Adenosine Triphosphate–Sensitive Potassium Channel Openers on Lung PreservationDepartment of Thoracic Surgery, Otsu Red-Cross Hospital, Otsu-City; and Department of Thoracic Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan Correspondence and requests for reprints should be addressed to Tatsuo Fukuse, M.D., Department of Thoracic Surgery, Otsu Red-Cross Hospital, 1-1-35 Nagara, Otsu-City 520-8511, Japan. E-mail: tafukuse{at}f4.dion.ne.jp
ATP-sensitive potassium channel (KATP) openers have been proven to be involved in ischemic preconditioning, which protects ischemic tissue. However, the effect of KATP openers on ischemia–reperfusion injury of the lungs remains unknown. We investigated whether a KATP opener, pinacidil, can attenuate ischemia–reperfusion injury using an ex vivo rat lung model. Heart–lung blocks were flushed and preserved with phosphate-buffered saline (control group) or with one of the solutions containing pinacidil (pinacidil group) or pinacidil + glibenclamide (a KATP blocker) (glibenclamide group). The control and glibenclamide groups showed significantly higher values with respect to shunt fraction, pulmonary arterial pressure, and peak inspiratory pressure than the pinacidil group. The concentrations of total adenine nucleotides and ATP in the lung after reperfusion became significantly lower in the control and glibenclamide groups than in the fresh group. Lipid peroxidation of the lungs increased significantly in the control and glibenclamide groups after reperfusion. State 3 mitochondrial respiration and State 3/4 ratios of mitochondrial respiration were significantly decreased in the lungs of the control and glibenclamide groups. These findings suggested that KATP openers would maintain the mitochondrial respiratory function during lung preservation, prevent lipid peroxidation after reperfusion, and attenuate ischemia–reperfusion injury.
Key Words: ATP-sensitive potassium channel openers • lung preservation • reperfusion injury • mitochondria • ATP
In clinical lung transplantation, graft failure occurs in 10–20% of cases, and this problem remains to be solved (1). Many studies have been performed on lung reperfusion injury. The involvement of reactive oxygen species, calcium paradox, energy depletion, mitochondrial dysfunction, and cytokines has been suggested, but the precise mechanism is still unclear (2). Recently, ATP-sensitive potassium channel (KATP) openers have been proven to be effective in the ischemic damage of the heart, and this channel has been demonstrated to be a key component of ischemic preconditioning that produces a marked protection against ischemic insult (3, 4). Several proposals have been suggested for the underlying mechanisms: reduced contraction by shortening the action potential duration to minimize the metabolic requirements of the ischemic cells and suppression of calcium ion entry into the cells by hyperpolarization of the cell membrane (5, 6). In addition, it is known that KATP openers suppress the release of superoxide from neutrophils (7). KATP openers are now considered to play central roles in the suppression of cardiac muscle reperfusion injury after it was clarified that the KATP exist not only on the cell membrane but also on the inner membrane of the mitochondria and that they play a role in the mitochondrial volume and energetics (8–10). On the other hand, it has been reported that opening of KATP causes an increased permeability in frog mesentery venules as measured by hydraulic conductivity (11, 12). However, there have been only a few studies on the effect of KATP openers on lung reperfusion injury (13, 14). Moreover, there has been no study concerning the effectiveness of pinacidil (a relatively pure KATP opener) in lung preservation. For example, Khimenko and coworkers (13) clearly showed that the activation of KATP could both protect against and reverse the endothelial damage associated with reperfusion injury after warm ischemia. However, in clinical lung transplantation, the donor lung is flushed and preserved with cold solution and perfused with blood when reperfused; therefore, the experimental setting of cold ischemia, which was used in our study, would be more practical for lung preservation. Yamashita and associates (14) reported the effect of nicorandil on reperfusion injury after cold ischemia in a canine lung transplantation model. However, nicorandil is a hybrid between a nicotinamide nitrate and a KATP opener, and the nitric oxide donor effect of nicorandil cannot be ignored. Nevertheless, a recent study reported the successful ischemic preconditioning of the lungs (15), which suggests that KATP openers are effective in lung preservation. Our previous study demonstrated the importance of vascular endothelial cell protection during lung preservation (16, 17). We also reported that the postreperfusion recovery of ATP levels was correlated with the lung reperfusion injury (18) and that mitochondrial dysfunction was likely to be involved in the lung reperfusion injury (19). To clarify the effectiveness of KATP openers in lung preservation, we examined the effect of pinacidil on pulmonary function after reperfusion. Furthermore, we studied the influence of this KATP opener on the energy metabolism during preservation and on the mitochondrial function after preservation.
Male Lewis rats (250–300 g) were used. The perfusion circuit was started using 30 ml of fresh blood that was obtained from three rats. The blood was perfused from the venous blood reservoir via pulmonary artery to the left lung using a double head roller pump. The laboratory animals were randomly divided into the following five groups. In the fresh group (n = 6), the heart–left lung blocks were reperfused immediately. In the other four groups, the heart–left lung blocks were preserved for 9 hours and then reperfused for measurement of the pulmonary function. In the control group (n = 6), 0.5 ml of phosphate-buffered saline (PBS) was administered intravenously 5 minutes before the flushing. Then, the samples were flushed with 50 ml of PBS and were inflated at an airway pressure of 14 cm H2O. In the pinacidil group (n = 6), 0.5 ml of PBS containing 0.5 mg of pinacidil was administered intravenously 5 minutes before the flushing. In the glibenclamide group (n = 6), 0.5 ml of PBS containing 0.25 mg of glibenclamide was administered intravenously 5 minutes before the flushing; then the samples were flushed with 50 ml of PBS containing 100 µM of pinacidil and 10 µM of glibenclamide. Pinacidil and glibenclamide were dissolved in dimethyl sulfoxide (DMSO) at concentrations of 1 mg/40 µl and 1 mg/100 µl, respectively. To determine the effect of DMSO on the ischemic cells, DMSO in PBS at the same concentrations as in the pinacidil group and in the glibenclamide group was administered intravenously, and then the samples were flushed likewise (the vehicle group). Blood gases in the effluent blood from the deoxygenator lung were analyzed immediately before reperfusion (baseline) and after reperfusion (at 10, 20, 30, 40, 50, 60, 90, and 120 minutes), and the wet-to-dry ratio (W/D ratio) was calculated. For the thiobarbituric acid reactants (TBARS) assay, we used the mediastinal lobe of the right lung immediately after flushing, the right middle lobe 9 hours after preservation, and one-third of the tissue of the left lung after completion of reperfusion (20). The concentration of adenine nucleotides was measured in the right mediastinal lobe immediately after flushing, in the right middle lobe after storage, and in the upper one-third of the left lung after completion of reperfusion (21, 22). Mitochondrial respiration was measured in each of the fresh, control, pinacidil, glibenclamide, and vehicle groups (n = 6, each). Mitochondria were isolated from the stored lung according to the modified Fisher's method (23). Oxygen use was measured polarographically with a Clark-type electrode. The protein concentrations were measured by the method of Lowry and colleagues (24). The terminology for mitochondrial respiratory states as defined by Chance and Williams (25) was employed. All values given in the text and figures are expressed as means ± standard error of the mean (SEM). The significance of differences among the five groups was determined using the standard analysis of variance procedures and Scheffe's multiple comparison test. A p value less than 0.05 was considered statistically significant.
In the present study, no experiment was excluded from the data due to technical errors. A total of 180 rats was used for this study as follows: deoxygenator lung, 30; bleeder rat, 90; experimental rat for study lung, 30; and mitochondrial respiration measurement, 30. The mixed venous blood was stable after reperfusion, and its pH, partial oxygen pressure in mixed venous blood (PvO2), and mixed venous CO2 pressure (PvCO2) were within the physiologic ranges. One of the six rats in each of the vehicle, control, and glibenclamide groups had a problem with ventilation of the experimental lungs due to exudation of fluid into the tracheal tube resulting from pulmonary edema within 60 minutes, and the measurements were stopped. However, all rats in the other two groups (i.e., the fresh and pinacidil groups) were used for measurement throughout the 120 minutes of reperfusion.
Shunt Fraction
Peak Inspiratory Pressure The peak inspiratory pressure (Figure 2) in the fresh and pinacidil groups was almost equal and stable throughout reperfusion. The PIP in the control, vehicle, and glibenclamide groups increased after reperfusion and was significantly greater than that in the pinacidil and fresh groups.
Pulmonary Arterial Pressure The pulmonary arterial pressure (Ppa) (Figure 1) in the fresh and pinacidil groups was stable throughout the observation period. The Ppa in the control, vehicle, and glibenclamide groups was significantly greater than that in the pinacidil and fresh groups throughout 120 minutes of reperfusion.
Wet-to-Dry Ratio
Adenine Nucleotides
Lipid Peroxidation There were no significant differences in the level of lipid peroxidation (Figure 4) among the groups after flushing and preservation. The level of lipid peroxidation in the control, vehicle, and glibenclamide groups increased after reperfusion and was significantly higher than that in the fresh and pinacidil groups.
Mitochondrial Respiration There was no significant decrease in the mitochondrial respiration (Table 1) in the pinacidil group after 9 hours of preservation. However, the State 3 respiration and the State 3/4 ratio in the control, vehicle, and glibenclamide groups were significantly lower than those in the fresh and pinacidil groups.
In this study, we have demonstrated that administration of KATP openers suppressed the reperfusion injury after lung preservation. Five structurally different types of potassium channel openers have been reported, i.e., cromakalim-type, pinacidil-type, minoxidil-type, aprikalim-type, and diazoxide-type (26). The common features among KATP openers include a hydrophobic group, an electron-deficient aromatic ring, and a hydrogen-binding site (27). Manley and coworkers reported that all these compounds interact with the same target protein but that the several different allosterically coupled receptor-binding sites are probably involved (26). Cromakalim and pinacidil have been the most studied KATP openers, and pinacidil, especially, has been used clinically for hypertension as an orally administered drug (28). Previously, Khimenko and coworkers (13) clearly showed that the activation of KATP (administration of cromakalim) could both protect against and reverse the endothelial damage associated with reperfusion injury after warm ischemia. Yamashita and coworkers (14) reported the effect of nicorandil, a hybrid between a nicotinamide nitrate and a KATP opener, on reperfusion injury after cold ischemia in a canine lung transplantation model. The present study is, to the best of our knowledge, the first to show the effectiveness of pinacidil, a relatively pure KATP opener, in cold storage of the lung. We also found that pinacidil maintained the mitochondrial respiratory function during preservation and that this effect was lost by a KATP blocker, glibenclamide. We reported previously that during lung preservation, the degree of morphologic injury in the mitochondria correlated with the levels of pulmonary dysfunction such as impairment of the lung oxygenation capacity and lung compliance as well as with the level of pulmonary edema (19). We also reported that the degree of mitochondrial morphologic injury became more severe with prolongation of the preservation time and that the concentrations of ATP and TAN decreased after reperfusion (18). Therefore, maintenance of the mitochondrial respiratory function by pinacidil during ischemia may play a major role in the amelioration of reperfusion injury. The recovery of the ATP and TAN levels in the pinacidil group after reperfusion also supports our proposal. It is possible that rupture of the cellular membrane led to the loss of the TAN and ATP in the cells. However, the TAN and ATP concentrations in the blood were similar in all groups. Therefore, the latter hypothesis is not probable. The present findings are also consistent with those of Monticello and coworkers (4) who reported that KATP openers attenuated the mitochondrial swelling with disrupted cristae and electron-dense deposits after preservation. In addition, Kobara and coworkers (29) suggested that the increase in ATP concentration after reperfusion during the ischemic preconditioning was caused by protection of the mitochondrial ATPase rather than by maintenance of the stored ATP to prevent ischemia–reperfusion injury. Because the presence of mitochondrial KATP in the pulmonary artery has not yet been reported, it is unknown whether the improvement in the mitochondrial function by KATP openers in the present study was caused by maintenance of the cellular homeostasis via the sarcolemmal KATP or by the direct role of KATP on the mitochondrial inner membrane. On the other hand, He and Curry (11, 12) reported that opening of KATP caused an increased permeability in frog mesentery venules as measured by hydraulic conductivity. It has also been reported that KATP have different electrophysiologic effects depending on the kind of animals (i.e., guinea pig versus rat) (30, 31) and vessels (mesenteric artery versus cerebral artery) (32). KATP openers have an effect of vasodilation on pulmonary artery in humans (33) and rats (34). In the present study, pinacidil improved pulmonary function and attenuated pulmonary edema. Khimenko and coworkers (13) also showed that KATP openers protected lung endothelium from ischemia–reperfusion injury in rats. Because we did not measure endothelial permeability or capillary pressure, the damage reversing effect of KATP openers remains unclear. Further investigations are required to clarify this issue. It has been reported that KATP openers suppress superoxide anions produced by neutrophils (7). The present study also demonstrated that administration of pinacidil prevented lipid peroxidation after reperfusion. As the main sources of reactive oxygen species after reperfusion, the xanthine–xanthine oxidase reaction, myeloperoxidase of the activated neutrophils, and mitochondrial respiration are likely to be involved (35). Although we did not measure myeloperoxidase activity, no significant differences in the neutrophil infiltration after reperfusion were observed in the lung tissue by light microscopy (data not shown). In addition, the possibility that pinacidil in the storage solution had an effect on the neutrophils can be ignored as pinacidil was not administered after reperfusion. Park and coworkers (36) reported that ischemic preconditioning reduced superoxide production and prevented impairment of the State 3 mitochondrial respiration induced by ischemia and reperfusion. In the present study, the mitochondrial respiratory function decreased in the control group but was maintained in the pinacidil group after preservation, thus indicating that the upregulated production of superoxides through the transition of mitochondrial permeability was likely to be responsible for the increased lipid peroxidation. In summary, we proved that KATP openers attenuated the reperfusion injury during lung preservation, maintained the mitochondrial respiratory function after preservation, and decreased lipid peroxidation after reperfusion. Therefore, KATP openers can be a useful tool in suppressing lung reperfusion injury. It is still unknown whether this mechanism of action is mediated via the sarcolemmal or mitochondrial KATP or via an action not only on the endothelial cells but also on the neutrophils. Further investigations are required to elucidate the precise mechanism of action.
This work was partially supported by a Grant-in-Aid (10671250) for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form December 7, 2000; accepted in final form March 22, 2002
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p < 0.01 versus the control, vehicle, and glibenclamide groups.
