Activation of endothelial NADPH oxidase during normoxic lung ischemia is KATP channel dependent
Qunwei Zhang,
Ikuo Matsuzaki,
Shampa Chatterjee, and
Aron B. Fisher
Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania
Submitted 10 May 2005
; accepted in final form 4 July 2005
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ABSTRACT
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Previous studies have shown endothelial cell membrane depolarization and generation of reactive oxygen species (ROS) in endothelial cells with abrupt reduction in shear stress (ischemia). This study evaluated the role of ATP-sensitive potassium (KATP) channels and NADPH oxidase in the ischemic response by using Kir6.2/ and gp91phox/ mice. To evaluate ROS generation, we subjected isolated perfused mouse lungs labeled with 2',7'-dichlorodihydrofluorescein (DCF), hydroethidine (HE), or diphenyl-1-pyrenylphosphine (DPPP) to control perfusion followed by global ischemia. In wild-type C57BL/6J mice, imaging of subpleural endothelial cells showed a time-dependent increase in intensity for all three fluorescence probes with ischemia, which was blocked by preperfusion with cromakalim (a KATP channel agonist) or diphenyleneiodonium (DPI, a flavoprotein inhibitor). Endothelial cell fluorescence with bis-oxonol, a membrane potential probe, increased during lung ischemia indicating cell membrane depolarization. The change in membrane potential with ischemia in lungs of gp91phox/ mice was similar to wild type, but ROS generation did not occur. Lungs from Kir6.2/ showed marked attenuation of the change in both membrane potential and ROS production. Thus membrane depolarization during lung ischemia requires the presence of a KATP channel and is required for activation of NADPH oxidase and endothelial ROS generation.
fluorescence microscopy; gp91phox; Kir6.2; mechanotransduction; membrane depolarization; perfused lung; reactive oxygen species; shear stress; adenosine 5'-triphosphate-sensitive potassium channel; reduced nicotinamide adenine dinucleotide phosphate
LUNG INJURY ASSOCIATED with ischemia-reperfusion (I/R) can result from lung transplantation, pulmonary artery thromboendarterectomy, cardiopulmonary bypass surgery, or other events associated with reversible obstruction of blood flow. This injury is manifested by nonspecific alveolar damage, lung edema, and increased blood flow resistance. During the past two decades, there has been a significant increase in our knowledge of the mechanism for tissue injury with I/R, and many studies have demonstrated that reactive oxygen species (ROS) play an important role (16, 17, 20). In systemic organs such as liver, intestine, kidney, heart, brain, and skeletal muscle, the major manifestations of tissue ischemia include cellular anoxia due to inadequate oxygen delivery and secondary acidosis; reoxygenation upon reperfusion results in a burst of ROS production that can result in oxidative tissue injury (11, 20). However, the lung responds differently because ischemia such as that which accompanies vascular occlusion does not compromise lung ventilation or oxygen delivery and therefore would not result in tissue anoxia or acidosis (14, 31). Hence, in the lung, the mechanism of injury resulting from ischemia can be distinguished from the effects of anoxia-reoxygenation (31).
Endothelial cells are highly sensitive to physical forces associated with blood flow (shear stress) and are able to transform these mechanical forces into electrical and biochemical signals (mechanotransduction). The response to altered shear includes modulation of membrane proteins and ion channels, activation of transcription factors and cognate target genes, and cellular reorganization with change in cell shape (7, 12). Some responses occur within seconds, whereas others require hours before a measurable adaptive change is apparent. Thus we postulated that altered shear stress associated with ischemia provides a signal for cellular response.
Previous studies in our laboratory using the isolated perfused rat lung and in vitro cell culture models indicated an increase in endothelial ROS generation during the ischemic period (1, 2, 14, 18, 30). Our previous results also showed that cessation of flow resulted in partial depolarization of the endothelial cell membrane preceding the increased generation of ROS (3, 4, 18, 25). Perfusion of lungs with cromakalim [an ATP-sensitive potassium (KATP) channel agonist] blocked both membrane depolarization and ROS generation (4, 25). We postulated that activation of KATP channels prevented channel closure associated with acute loss of shear stress. ROS production also was inhibited by the flavoprotein inhibitor diphenyleneiodonium chloride (DPI), which has been widely used as an inhibitor of NADPH oxidase (2, 31). Therefore, we propose that loss of the mechanical component of flow during lung ischemia results in endothelial cell membrane depolarization and the activation of NADPH oxidase.
The present study investigated endothelial cell membrane depolarization and ROS production during lung ischemia using gene-targeted mice deficient in inwardly rectifying potassium channel (Kir) 6.2 or gp91phox. Kir6.2 is the pore-forming subunit of the endothelial KATP channel, and gp91phox is the flavoprotein component of the phagocyte-type NADPH oxidase. The results provide evidence for understanding the role of KATP channels and NADPH oxidase in the endothelial cell response to lung ischemia.
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MATERIALS AND METHODS
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Chemicals and reagents.
Bis-(1,3-dibutylbarbituric acid) trimethine oxonol (bis-oxonol), dimethyl sulfoxide (DMSO), diphenyl-1-pyrenylphosphine (DPPP), DiI-acetylated low-density lipoprotein (DiI-AcLDL), hydroethidine (HE), and dichlorodihydrofluorescein diacetate (H2DCF) were obtained from Molecular Probes (Eugene, OR). Cromakalim and Trolox (a water-soluble form of vitamin E) were purchased from Sigma Chemical (St. Louis, MO). DPI was from Calbiochem (La Jolla, CA). Catalase was from Boehringer Mannheim (Indianapolis, IN). All chemicals used were of analytical grade.
Animals and isolated cells.
Animal use was reviewed and approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Male C57BL/6J, Tie-2 GFP, and gp91phox gene-targeted mice weighing
20 g were obtained from Jackson Labs (Bar Harbor, ME). Tie-2 GFP mice genetically express green fluorescent protein (GFP) only in endothelium. Breeding pairs of mice deficient in KATP channels generated by targeted disruption of the Kir6.2 gene were obtained from Chiba University (21) and bred in the University of Pennsylvania animal facilities. Kir6.2 gene-targeted mice had been backcrossed for five generations to the C57BL/6J background.
Pulmonary microvascular endothelial cells were isolated from mouse lungs by modification of a previously described procedure (13). In brief, mouse lungs were cleared of blood, minced finely, incubated with 0.1% collagenase at 37°C, and filtered through a sterile 100-µm cell strainer to remove macrophages. The cell suspension was incubated with platelet endothelial cell adhesion molecule (PECAM) antibody (rat anti-mouse CD31; BD Biosciences, San Diego, CA) added to the cell suspension, and endothelial cells were isolated by magnetic bead separation using sheep anti-rat IgG coated Dynabeads (Dynal Biotech, Oslo, Norway). The Dynabeads with attached cells were seeded onto gelatin-coated flasks and allowed to grow to 80% confluence before use. We confirmed the endothelial phenotype of the preparation by demonstrating cellular uptake of DiI-Ac-LDL and reactivity to anti-PECAM-1 (9).
RNA isolation.
Total RNA from mouse pulmonary microvascular endothelial cells (MPMVEC) (
80% confluent) was obtained by addition of 2 ml of TRIzol (Life Technologies, Gaithersburg, MD) directly to the flask, incubation for 5 min at room temperature, and addition of 0.2 ml of chloroform per 1 ml of TRIzol. After vigorous mixing and centrifugation, we separated the colorless upper aqueous phase, and total RNA was precipitated by addition of 0.5 ml of isopropyl alcohol per ml TRIzol used for the initial homogenization. After being washed with 75% ethanol, the RNA pellet was dissolved in 100 µl of RNase-free H2O. The RNA concentration was measured at an optical density at 260 nm using a DU 640B Spectrophotometer (Beckman Coulter, Fullerton, CA).
RT-PCR.
The presence of KATP channel mRNA in mouse lung endothelial cells was determined by PCR. In brief, 1 µg of total RNA was reverse-transcribed into cDNA by using the 1st Strand cDNA Synthesis Kit for RT-PCR (Roche). PCR was performed on a Mastercycler (Eppendorf, Westbury, NY) using 35 cycles at 94°C for 30 s, at 58°C for 45 s, and at 72°C for 45 s for Kir6.1 and Kir6.2. For
-actin, we used 28 cycles for 30 s at each of the three temperatures. The primers were 5'-TATCATACAGGGGGCTACGC-3' and 5'-GTCTTCTAGGAGGACGCGTG-3' for mouse Kir6.1. The primers for Kir6.2 were 5'-AAGAAAGGCAACTGCAACGT-3' and 5'-CCCCATAGAATCTCGTCAGC-3'. The primers for mouse
-actin were 5'-GGCATTGTTACCAACTGGGAC-3' and 5'-ACCAGAGGCATACAGGGACAG-3'. The PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining.
Isolated lung perfusion.
The isolated perfused lung technique used for this study has been described previously for rat lungs (14, 25) and was slightly modified for perfusion of mouse lungs. Briefly, mice were anesthetized with 50 mg/kg intraperitoneal sodium pentobarbital and continuously ventilated through a tracheal cannula with 5% CO2 in air (BOC group, Murray Hill, NJ). The chest was opened, and the pulmonary circulation was cleared of blood by gravity flow of perfusion through a cannula inserted in the main pulmonary artery, exiting from the transected left ventricle. The perfusate was Krebs-Ringer bicarbonate solution (KRB, in mM: 118.45 NaCl, 4.74 KCl, 1.17 MgSO4·7H2O, 1.18 KH2PO4, and 24.87 NaHCO3) plus 10 mM glucose and 5% dextran to maintain isotonicity. The heart-lung preparation was dissected en bloc and placed onto a 48 x 60 x 0.16-mm cover glass in a specially designed Plexiglas chamber with ports for tracheal and pulmonary artery cannulas. The cardiovascular ports were connected to a peristaltic pump that recirculated 30 ml of perfusate at a constant flow rate of 2 ml/min through the vascular bed. The heart muscle was trimmed away, and a local anesthetic (0.05 mg xylazine) was injected subepicardially into the posterior wall of the right atrium to abolish lung movement artifact due to contraction of the remaining cardiac muscle. The isolated perfused lung was placed on the stage of an inverted microscope for imaging studies of the subpleural vasculature using reporter fluorophores. Isolated lungs were shaded from direct light during the perfusion experiments. Global ischemia was produced by the abrupt discontinuation of perfusion.
Endothelial ROS generation and lipid peroxidation.
Dichlorofluorescein (DCF) fluorescence was used to monitor generation of oxidants with lung ischemia (2, 4, 31). Nonfluorescent H2DCF diacetate is converted by intracellular esterases to H2DCF, which serves as a substrate for intracellular oxidants to generate highly fluorescent DCF. A 5 mM stock solution of H2DCF diacetate was prepared in 100% ethanol, stored under N2 at 20°C in the dark, and added to the lung perfusate to make a final concentration of 5 µM. HE fluorescence also was used to monitor intracellular generation of ROS during nonhypoxic lung ischemia (1). HE, an uncharged dye that readily permeates the cell membrane, is converted to a fluorescent ethidium congener on oxidation (32). A 50 mM stock solution of HE was prepared in DMSO, stored under N2 at 20°C in the dark, and added to the lung perfusate to make a final concentration of 10 µM. Lipid peroxidation associated with ROS generation was evaluated with DPPP, a hydrophobic dye that yields a fluorescent product by its interaction with lipid hydroperoxides (19, 23). DPPP was solubilized in DMSO, as a 10 mM stock solution, stored at 20°C in darkness, and added to the perfusate to give a final concentration of 10 µm.
Endothelial cell membrane potential.
Membrane potential was determined with bis-oxonol (3). This dye, which is nonfluorescent in solution, localizes to the plasma membrane and increases in fluorescence intensity with membrane depolarization. Endothelial depolarization by perfusion with increased K+-containing KRB was used to provide an approximate calibration for the bis-oxonol fluorescence signal. Lungs were loaded with bis-oxonol dye, perfused under normal conditions with KRB (5.9 mM K+) to get baseline fluorescence, and then abruptly changed to KRB solutions containing elevated K+ concentrations. Na+ in the high K+ solutions was reduced equivalently to maintain isosmolality.
Intravital subpleural microvascular endothelial cell microscopy.
Intravital microscopy was performed as described previously for the rat lung (2, 4, 19, 25, 29). Briefly, the chamber was placed on the stage of an epifluorescence microscope fitted with a x60 objective (Nikon Diaphot TMD) and equipped with an optical filter changer (Lambda 102; Sutter Instrument, Novato, CA), a Hamamatsu ORCA-100 digital camera (Hamamatsu, Bridgewater, NJ), and MetaMorph imaging software (Universal Imaging, Downingtown, PA). Excitation of the lung surface was accomplished with a mercury lamp fiber-optic light source and the appropriate filter set as follows: for bis-oxonol, 480 ± 20 nm excitation, 505 LP dichroic, 535 ± 25 emission; for DPPP, 335 ± 10 nm excitation, 505 LP dichroic, 405 ± 20 nm emission; for DCF, 485 ± 5 nm excitation, HQ-41001b with 510 ± 10 nm emission; for HE, 480 nm excitation, 585 ± 25 nm emission; and DiI-AcLDL, 545 ± 15 nm excitation, 585 ± 25 nm emission. Images were obtained from subpleural vessels of
3050 µm in diameter.
For imaging studies, lungs were preperfused with bis-oxonol (100 nM), H2DCF (5 µM), HE (10 µm), or DPPP (10 µM) for 30 min to allow uptake of the fluorophore, and intravascular dye was then removed by 10-min perfusion with dye-free medium to reduce background fluorescence. In some experiments, cromakalim (30 µM), DPI (10 µM), catalase (1,000 U/ml), or Trolox (500 µM) was added to the dye loading solution during the preperfusion period. Images were acquired during a 10-min control period of continuous perfusion and then after the peristaltic pump was abruptly stopped (ischemia). Ventilation was stopped briefly (<15 s) to permit collection of fluorescence images from randomly selected areas of each lung. For quantitation, an area of interest containing one or more endothelial cells was outlined, and the percent change of ratio fluorescence intensity (% of baseline) was calculated. Values for five to seven areas of interest (endothelial cells) were averaged to obtain a mean value for each lung.
Statistical analysis.
Results are expressed as means ± SE generally for three to four lungs for each condition. For studies of time-dependent change, significance of parametric differences among groups was evaluated with two-way analysis of variance; if the F-value was significant, groups were then compared at each time by one-way analysis of variance followed by Dunnett's t-test. Statistical analyses were carried out using Sigma Stat (Jandel Scientific, San Raphael, CA). Differences were considered significant at P < 0.05.
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RESULTS
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KATP channel subunits in mouse lung endothelial cells.
KATP channels are composed of a pore-forming subunit, either Kir6.1 or 6.2. We have shown previously the presence of Kir6.2 in rat pulmonary microvascular endothelial cells (8). The expression of Kir6.1 and Kir6.2 mRNA was evaluated by RT-PCR in MPMVEC. Kir6.2 was detected in wild-type and gp91phox/ MPMVEC but was not present in Kir6.2/ MPMVEC (Fig. 1). Kir6.1 mRNA was not detected in any of the MPMVEC lines.

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Fig. 1. Identification of ATP-sensitive potassium (KATP) channel subunits in mouse lung microvascular endothelial cells. Total RNA was isolated from wild-type (WT), inwardly rectifying potassium channel (Kir) 6.2/, and gp91phox/ mouse lung microvascular endothelial cells in monolayer culture. The expression of mRNA for Kir6.1 and Kir6.2 (left) and -actin (right) was detected by reverse transcription PCR and visualized on a 2% agarose gel stained with ethidium. Lanes 1 and 4, gp91phox/; lanes 2 and 5, Kir6.2/; lanes 3 and 6, WT. DNA molecular markers (lane 7) were used to confirm the size of the PCR products.
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Endothelial cell identification in situ.
DiI-AcLDL was used as a marker for endothelial cells. Lungs perfused for 30 min with this fluorophore showed cellular uptake consistent with microvascular endothelial cells (Fig. 2A). This pattern of labeling is similar to that previously described for the rat lung (2). The pattern for DCF fluorescence was similar and colocalized with DiI-AcLDL. The endothelial localization of fluorophores also was evaluated with lungs from Tie-2 GFP mice, which showed colocalization of GFP and HE (Fig. 2B).

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Fig. 2. Identification of microvascular endothelium in situ. In situ imaging of lungs to evaluate colocalization of 2',7'-dichlorodihydrofluorescein (DCF) and hydroethidine (HE) with endothelial cell markers. A: colocalization of DCF and DiI-acetylated low-density lipoprotein (DiI-AcLDL). Isolated mouse lungs were perfused with 50 µg of DiI-AcLDL and 5 µM dichlorodihydrofluorescein diacetate (H2DCF-DA) for 30 min and then placed on a microscope stage for in situ imaging of subpleural alveoli. Images from the same area were taken at excitation/emission of 485/510 nm for DCF (left) and 545/585 nm for DiI-AcLDL (center). The images were largely colocalized (right). Alv, alveolar space; End, microvascular endothelium. B: colocalization of HE and green fluorescent protein (GFP). Isolated lungs from Tie-2 GFP mouse were perfused with 10 µM HE for 30 min and then placed on a microscope stage for in situ imaging of subpleural alveoli. Images from the same area were taken at excitation/emission of 545/585 nm for GFP (left) and 480/585 nm for HE (center). The images were largely colocalized (right).
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ROS generation during lung ischemia.
Imaging of subpleural microvessels for DCF and HE fluorescence was used to detect ROS generation by mouse lung endothelium. There was essentially no change in fluorescence during a 15-min period of continuous (control) perfusion (images not shown), but fluorescence increased progressively with cessation of flow in wild-type mouse lungs (Figs. 3A and 4A). The increase in fluorescence with stop flow was statistically different from continuous perfusion at 1 min and at all subsequent time points (Figs. 3B and 4B). The increase in DCF fluorescence with ischemia was abolished by pretreatment of lungs with catalase and was markedly attenuated by pretreatment of lungs with the KATP channel agonist cromakalim or the flavoprotein inhibitor DPI (Fig. 3B). Cromakalim also inhibited the increase in HE fluorescence with ischemia (Fig. 4B). Unlike the wild-type lungs, DCF and HE fluorescence were unchanged from baseline with ischemia in lungs from gp91phox/ mice (Figs. 3B and 4B). In Kir6.2/ mice, fluorescence showed a delayed increase with ischemia that did not become significant until 11 min for DCF and 5 min for HE; the change in fluorescence was significantly depressed by
50% compared with lungs from wild-type mice (Figs. 3B and 4B).

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Fig. 3. DCF fluorescence in subpleural endothelial cells in the intact mouse lung. Lungs were preperfused with H2DCF-DA for 30 min followed by dye-free perfusate. Images were acquired at 1-min intervals for 15 min after global cessation of flow (ischemia). The time below the images indicates minutes following cessation of flow to the lung. All images were acquired with the same gain settings. Images are in pseudocolor with intensity scale shown in A. A: imaging of endothelial response to ischemia in lungs of WT mouse. B: quantitation of time course of DCF fluorescence intensity change with lung ischemia. Mean ± SE change in fluorescence intensity of 34 lungs (each representing the average value for 36 endothelial cells) is plotted vs. time for continuously perfused (control) or ischemic lungs for WT, gp91phox/, and Kir6.2/ mice. The effect of catalase (1,000 U/ml), cromakalim (30 µM), and diphenyleneiodonium (DPI, 10 µM) is shown for WT mouse lungs. Inhibitors were added to the perfusate during the 30-min equilibration period. *P < 0.05 vs. the corresponding control lung; #P < 0.05 for ischemia in WT vs. Kir6.2/ mice.
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Fig. 4. HE fluorescence in subpleural endothelial cells in the intact mouse lungs. Lungs were preperfused with HE for 30 min of equilibration followed by dye-free perfusate. Images were acquired at 1-min intervals for 15 min after global cessation of flow (ischemia). The time below the image indicates minutes following cessation of flow to the lung. All images were acquired using the same gain settings. Images are in pseudocolor with intensity scale shown in A. A: images of endothelial response to ischemia in lungs of a WT mouse. B: quantitation of time course of HE fluorescence intensity change with lung ischemia. Mean ± SE change in fluorescence intensity of 34 lungs (each representing the average value for 36 endothelial cells) is plotted vs. time for continuously perfused (control) or ischemic lungs for WT, gp91phox/, and Kir6.2/ mice. Cromakalim (30 µM) was added to the perfusate during the equilibration period. *P < 0.05 vs. corresponding control lung; #P < 0.05 for ischemia in WT vs. Kir6.2/.
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Lipid peroxidation was monitored in real time by using DPPP as a fluorophore (19). Endothelial cells in situ in subpleural microvessels of wild-type mouse lungs showed no significant change in DPPP fluorescence during a 15-min period of continuous perfusion (images not shown). Abrupt cessation of flow (ischemia) resulted in increased DPPP fluorescence in wild-type mice (Fig. 5A) similar to that seen previously with rat lungs (19). The DPPP change compared with continuous flow was statistically significant at 4-min ischemia (Fig. 5B). The increase in DPPP fluorescence intensity with ischemia was completely blocked when lungs were preperfused with Trolox and in lungs from gp91phox/ mice (Fig. 5B). Trolox is a water-soluble form of vitamin E that partitions into cell membranes, protects against membrane lipid peroxidation, and has been shown previously in rat lungs to inhibit DPPP oxidation with ischemia (19, 24). Lungs from Kir6.2/ mice showed
50% of the change in DPPP fluorescence intensity with ischemia compared with that seen in the wild-type mice (Fig. 5B).

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Fig. 5. Diphenyl-1-pyrenylphosphine (DPPP) fluorescence in subpleural endothelial cell in the intact mouse lung. Lungs were preperfused with DPPP for 30 min followed by dye-free perfusate and then images were acquired at 1-min intervals for 15 min after global cessation of flow (ischemia). Time below the images indicates minutes following cessation of flow to the lung. All images were acquired using the same gain settings. Images are in pseudocolor with intensity scale shown in A. A: imaging of endothelial response to ischemia for WT mice. B: quantitation of time course of DPPP fluorescence intensity change with lung ischemia. Fluorescence intensity is plotted vs. time for continuously perfused (control) and ischemic lungs from WT, Trolox-treated (500 µM), gp91phox/, and Kir6.2/ mouse lungs. Values are means ± SE for n = 34. *P < 0.05 vs. the corresponding control lung; #P < 0.05 for ischemia in WT vs. all other conditions.
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Endothelial cell membrane potential during lung ischemia.
Change of endothelial cell membrane potential was measured by in situ imaging of bis-oxonol fluorescence. Ischemia in lungs from wild-type mice resulted in increased endothelial cell fluorescence intensity compatible with cell membrane depolarization (Fig. 6, A and B). Maximal change in fluorescence was observed in the first 2 min; the subsequent decrease from the peak value was not statistically significant (Fig. 6B). The depolarization response was blocked by pretreatment with cromakalim, the KATP channel agonist (Fig. 6B). The change in bis-oxonol fluorescence with ischemia in lungs from gp91phox/ mice was similar to the wild type (Fig. 6B). With lungs from Kir6.2/ mice, endothelial cell membrane potential with ischemia showed an attenuated response that was followed by a rapid return to baseline (Fig. 6B).

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Fig. 6. Bis-oxonol fluorescence in subpleural endothelial cells in the intact mouse lung. Lungs were preperfused with 100 nM bis-oxonol for 30 min followed by dye-free perfusate. Images were acquired at 1-min intervals for 5 min after global cessation of flow (ischemia). A: imaging of endothelial response to ischemia in lungs of a WT mouse. Lungs were subjected to abrupt cessation of flow at zero time. All images were acquired using the same gain settings. Images are in pseudocolor with intensity scale shown in A. B: quantitation of time course of bis-oxonol fluorescence intensity change with lung ischemia. Mean ± SE change in fluorescence intensity for 34 lungs is plotted vs. time for continuously perfused (control) or ischemic WT, gp91phox/, and Kir6.2/ lungs. Cromakalim (30 µM) was added during the 30-min preperfusion period. *P < 0.05 vs. control (continuous flow) at that time point for the same lung background. C: effect of high-K+ perfusion on bis-oxonol fluorescence from endothelial cells in the intact mouse lung. The bis-oxonol fluorescence signal was calibrated by perfusion of isolated lungs with increased K+ to induce membrane depolarization. Cells were loaded with bis-oxonol and perfused with KRB (5.9 mM K+) to obtain a baseline. The perfusate then was abruptly changed to modified KRB with 7.5, 10, or 24 mM K+ for WT lungs and 10 or 24 mM K+ for Kir6.2/ lungs. Values are means ± SE for 34 lungs under each condition.
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Perfusion with varying K+ was used to calibrate the results for cell membrane depolarization. Perfusion with high K+ (7.524 mM) in wild-type lung resulted in a K+ concentration-dependent increase in endothelial cell fluorescence intensity indicating decreased (i.e., a less negative) cell membrane potential (Fig. 6C). Bis-oxonol fluorescence increased
5% at 15 min after the start of perfusion with 10 mM K+ and increased 9.5% with 24 mM K+. Thus the depolarization response to ischemia is equivalent to perfusing lungs with
10 mM K+. If one assumes that the resting membrane potential is 60 mV, 10 mM K+ should cause a change in membrane potential of
14 mV. With lungs from Kir6.2/ mice, the change in bis-oxonol fluorescence with 24 mM K+ was slightly less than with wild-type lungs but was significantly less with 10 mM K+ (Fig. 6C).
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DISCUSSION
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Our laboratory has previously established an isolated perfused rat lung model that allowed us to separate ischemic from anoxic effects. In this model, ventilation of the lung is continued during the ischemic period so that tissue oxygenation and ATP content are unchanged and cellular responses are not the result of tissue hypoxia (3, 14, 31). Ischemia resulted in cell membrane depolarization and ROS generation in rat lung endothelial cells in situ (13, 25, 31), which was confirmed with in vitro cell culture models (18, 30). The presence of a KATP channel agonist, cromakalim (or the active enantiomer lemakalim), prevented the response to ischemia, suggesting that these channels were responsible for the response (4, 25).
For the present study, we utilized an isolated mouse lung preparation to investigate knockout mouse models that might provide mechanistic insights not otherwise attainable. The mouse models were knockout of the KATP channel, the putative channel responsible for membrane depolarization, and knockout of NADPH oxidase, the putative enzyme responsible for ROS generation. Imaging studies in the mouse lung showed that DCF colocalized with DiI-AcLDL, an endothelial cell marker, and that HE colocalized with GFP-stained endothelial cells. The pattern of localization for the other fluorophores (bis-oxonol, DPPP) was similar. Thus these models appear to be suitable for the investigation of the pulmonary response to altered perfusion.
Fluorescence imaging in the intact lung was used to evaluate ROS generation. The fluorescence probes used were H2DCF, HE, and DPPP. H2DCF is a nonspecific radical trap that is oxidized to DCF by H2O2 but is also sensitive to other radical species. DCF is susceptible to photoenhancement so that the observation period must be kept to a minimum. Increased DCF fluorescence in endothelial cells of intact lungs indicates the generation of ROS during lung ischemia; inhibition of DCF fluorescence increase by catalase indicates that DCF oxidation is associated with increased formation of H2O2, presumably by dismutation of O2·. HE reacts primarily with O2· to form a characteristic product that has recently been described as 2-hydroxyl ethidium (32). With the excitation/emission parameters used in this study, the presence of ethidium as a contaminant in HE preparations can result in high background fluorescence and partially obscure the signal associated with formation of the oxidation product (32). Increased HE fluorescence with ischemia suggests an intracellular source for O2·, although some permeation of O2· generated in the extracellular space by NADPH oxidase cannot be excluded. DPPP is a highly hydrophobic compound that localizes in membranes and is oxidized by lipid hydroperoxides to give a strongly fluorescent product (19, 23, 28). DPPP is oxidized to a lesser degree by other organic hydroperoxides but only weakly by H2O2 (19). The observation period for DPPP also must be kept to a minimum since this dye is very sensitive to photobleaching. Increased DPPP fluorescence during ischemia and its inhibition by Trolox indicate peroxidation of cellular (presumably membrane) lipids.
The three redox-sensitive probes all indicated increased ROS generation with ischemia. Increased ROS generation with ischemia was blocked when lungs were pretreated with DPI, a flavoprotein inhibitor that inhibits NADPH oxidase. We have previously demonstrated that DCF oxidation during 1-h ischemia in mouse lungs is abolished by gp91phox knockout (2). The present results in the gp91phox/ mouse provide data at early time points for DCF and HE oxidation in addition to measurement of DPPP fluorescence. These results showing loss of effect in lungs from mice with absent gp91phox confirm that NADPH oxidase is the ROS generator with lung ischemia.
The present study has demonstrated that cessation of flow results in endothelial cell membrane depolarization in the mouse lung, similar to the results of previous studies in the rat (2, 3, 25). Because depolarization is blocked by pretreatment with cromakalim, we have proposed that the ischemic effect is mediated by a KATP channel. This channel is present in many tissues, including cardiac myocytes (22), pancreatic
-cells (10), skeletal muscle (26), smooth muscle (27), brain (5), pituitary (6), and rat pulmonary microvascular endothelial cells (8). The KATP channel consists of two types of subunits: an inwardly rectifying pore-forming subunit (Kir6.1 or Kir6.2) and a regulatory sulfonylurea receptor subunit (15). We have shown previously that Kir6.2 is the KATP channel pore-forming subunit in rat pulmonary microvascular endothelial cells (8), and the present study demonstrates the same channel by PCR in endothelial cells of mouse lung. Kir6.1 mRNA was not found in lung endothelial cells. To study the role of KATP channels, Kir6.2/ mice were used to determine whether absence of the channel alters the endothelial cell response to lung ischemia. In the absence of Kir6.2, lungs showed a relatively small and transient depolarization response to ischemia. Thus Kir6.2 channels appear to mediate the effect of ischemia on pulmonary vascular endothelial cell membrane potential. Although it is likely that endothelial cell membrane potential is maintained by other channels in the Kir6.2/ mouse, those channels show a relatively weak response to the acute loss of shear. The effect of added external K+ suggests that the Kir6.2/ endothelial cells are relatively resistant to depolarization compared with the wild type. One caveat is important in the interpretation of the bis-oxonol fluorescence data. Because the results are presented as percent change, a highly negative resting membrane potential could dampen the fluorescence response to ischemia in the Kir6.2/ lungs. Although we have not been able to measure the resting endothelial cell membrane potential in the intact lung, it seems more likely that the resting potential is less rather than more negative in the Kir6.2/ cells. The channels responsible for the resting membrane potential in the Kir6.2/ endothelium have not been identified.
Inhibition of ROS generation with ischemia by the presence of cromakalim suggests that ROS generation is linked to membrane depolarization. In the Kir6.2/ mice, imaging of DCF and HE fluorescence indicated a significantly lower level of endothelial ROS generation during the initial 15 min of lung ischemia compared with the wild type and measurement of DPPP fluorescence indicated a lower level of lung lipid peroxidation. Thus the KATP channel appears to play a major role in endothelial ROS generation with ischemia, and this effect is mediated via membrane depolarization. The partial ROS response in Kir6.2/ mice may reflect partial membrane depolarization associated with other channels as discussed above or could be due to other mechanisms that are peculiar to the gene-targeted mouse. Although depolarization of the endothelial cell membrane with ischemia indicates that this pathway is sensitive to shear stress, it is still not clear whether the KATP channel itself is shear sensitive or is regulated by some other shear stress-sensitive protein.
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GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-60290 and HL-75587.
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ACKNOWLEDGMENTS
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We thank Drs. S. Seino and T. Miki for providing breeding pairs of Kir6.2/ mice, Dr. Y. Manevich for advice about DPPP, and J. Rossi for typing the manuscript.
Current address for I. Matsuzaki: Second Department of Surgery, Akita University School of Medicine, Hondo 1-1-1, Akita City, Japan 010-8543.
Presented in part at the Experimental Biology meeting in Washington, DC, April 2004.
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FOOTNOTES
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Address for reprint requests and other correspondence: A. B. Fisher, Inst. for Environmental Medicine, Univ. of Pennsylvania School of Medicine, 1 John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6068 (e-mail: abf{at}mail.med.upenn.edu)
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