Lack of amiloride-sensitive transport across alveolar and respiratory epithelium of iNOS(-/-) mice in vivo

Karin M. Hardiman1, J. Russell Lindsey2, and Sadis Matalon1,2,3

Departments of 1 Physiology and Biophysics, 2 Comparative Medicine, and 3 Anesthesiology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The extent to which endogenously generated nitric oxide alters Na+ transport across the mammalian alveolar epithelium in vivo has not been documented. Herein we measured alveolar fluid clearance and nasal potential differences in mice lacking the inducible form of nitric oxide synthase [iNOS; iNOS(-/-)] and their corresponding wild-type controls [iNOS(+/+)]. Alveolar fluid clearance values in iNOS(+/+) and iNOS(-/-) anesthetized mice with normal oxygenation and acid-base balance were ~30% of instilled fluid/30 min. In both groups of mice, fluid absorption was dependent on vectorial Na+ movement. Amiloride (1.5 mM) decreased alveolar fluid clearance in iNOS(+/+) mice by 61%, whereas forskolin (50 µM) increased alveolar fluid clearance by 55% by stimulating amiloride-insensitive pathways. Neither agent altered alveolar fluid clearance in iNOS(-/-) mice. Hyperoxia upregulated iNOS expression in iNOS(+/+) mice and decreased their amiloride-sensitive component of alveolar fluid clearance but had no effect on the corresponding values in iNOS(-/-) mice. Nasal potential difference measurements were consistent with alveolar fluid clearance in that both groups of mice had similar baseline values, which were amiloride sensitive in the iNOS(+/+) but not in the iNOS(-/-) mice. These data suggest that nitric oxide produced by iNOS under basal conditions plays an important role in regulating amiloride-sensitive Na+ channels in alveolar and airway epithelia.

nitric oxide; osmolality; hyperoxia; nasal potential difference; inducible nitric oxide synthase-deficient mice


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ESTABLISHED that alveolar epithelial cells are capable of transporting Na+ in a vectorial fashion both in vivo and in vitro (for reviews see Refs. 33 and 34). Na ions in the alveolar lining fluid passively diffuse down their electrochemical gradient into alveolar type II (ATII) cells through amiloride-sensitive channels located in their apical membranes (22, 50) and are extruded across the basolateral membranes by various subunits of the ouabain-sensitive Na+-K+-ATPase (8). K ions, which enter the ATII cell in exchange for Na+, exit the cell down their electrochemical gradient, through K+ channels located in the basolateral membrane. It is the active Na+ transport across the alveolar epithelium that creates an osmotic gradient that then drives fluid from the alveolar to the interstitial space. Various studies in humans and animals indicate that active Na+ reabsorption plays an important role in diminishing alveolar fluid and improving oxygenation in both patients with cardiogenic and noncardiogenic edema (35) and in animals with hyperoxic lung injury (38).

Because of their location, alveolar epithelial cells are often exposed to increased intracellular and extracellular concentrations of reactive oxygen and nitrogen species. Most mammalian cells have the capacity to produce nitric oxide (NO) via the oxidative deamination of L-arginine by either the Ca2+-sensitive (c) or the Ca2+-insensitive (i) isoform of nitric oxide synthase (NOS). Pertinent sources of pulmonary NO include activated macrophages (20) and airway (41) and ATII cells (40). Consequently, in many pathological conditions, there may be significant levels of NO present in the alveolar lining fluid and thus in close proximity to alveolar epithelial cells. The biological effects of NO depend on its concentration, the biochemical composition of the target, and the presence of other radicals. NO may bind to the heme group of soluble guanylate cyclase, resulting in increased cellular cGMP levels (19); it may react with superoxide at diffusion-limited rates (6.7 × 109 M-1 · s-1) to produce peroxynitrite (1) and higher oxides of nitrogen, or, in the presence of an electron acceptor, it may react with thiols to form nitrosothiols (15). Existing evidence in the literature and our own data indicate that various redox states of NO modify the activity of both cation channel proteins and the Na+-K+-ATPase either by altering signal transduction pathways, by oxidizing or nitrating ion-transporting proteins directly, or by affecting the cytoskeleton (5, 14, 17, 21, 26).

However, the extent to which endogenously generated NO alters Na+ transport across the mammalian alveolar epithelium has not been elucidated. This is an important question since, as mentioned above, a number of inflammatory agents are known to upregulate NO production by both inflammatory and alveolar epithelial cells in pathological situations. Furthermore, NO generation by either cNOS or iNOS under baseline conditions may regulate the activity of amiloride-sensitive ion channels in lung epithelial cells. For example, it has been proposed that the lack of iNOS in the airways of mice with cystic fibrosis (25) may contribute to the lack of cAMP-stimulated chloride secretion and the increase in the rate of amiloride-sensitive Na+ transport (7).

Herein we measured levels of amiloride-sensitive alveolar fluid clearance (AFC) in the lungs of C57BL/6 iNOS-deficient [iNOS(-/-)] mice, which have normal phenotype except for decreased ability to clear pathogens (16), and age- and genetic-matched C57BL/6 wild-type control [iNOS(+/+)] mice by instilling into their lungs isosmotic fluid containing 5% BSA and measuring changes in BSA concentration over a 30-min period. We then repeated these measurements in mice that were exposed to 100% O2 at one atmosphere for 55 h, which upregulated iNOS levels in the inflammatory cells in the lungs of iNOS(+/+) mice. The effects of hyperoxia on the lungs of these mice were measured by quantitative histopathology. We also measured potential difference across the nasal airways of iNOS(+/+) and iNOS(-/-) mice in the presence and absence of amiloride. Because our data indicate the absence of amiloride-sensitive Na+ transport across both the alveolar epithelium and airways of iNOS(-/-) mice, we measured fluid clearance with a Na+-free solution to determine if AFC was the result of active Na+ transport, and we tried to stimulate amiloride-sensitive fluid clearance by adding forskolin to the instillate. In contrast to previous studies in which AFC was measured in situ (12), in isolated perfused lungs (18), or in ventilated mice with unknown acid-base status (11), all of our measurements were performed in living, ventilated, anesthetized mice with adequate oxygenation and acid-base balance. Results presented here indicate that iNOS(-/-) mice have normal levels of basal AFC, dependent on vectorial transport of Na+ moving across alveolar epithelial cells through amiloride-insensitive pathways that are not stimulated by an increase in cAMP.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

Animals. Pathogen-free C57BL/6NCr [iNOS(+/+)] mice were obtained from Frederick Cancer Research and Development Center (National Cancer Institute, Frederick, MD). Breeding pairs of C57BL/6J-NOS2tm1Lau mice [iNOS(-/-)] were obtained from Jackson Laboratory (Bar Harbor, ME). Mice were bred and maintained in autoclaved microisolator cages (Lab Products, Maywood, NJ) and were provided with food (Teklad, Madison, WI) and water ad libitum. All mice used in studies were 8-14 wk old and 20-30 g in weight. Routine surveillance studies in these mice, performed by one of us (J. R. Lindsey) as previously described (10), showed that these mice were negative for significant murine pathogens.

Exposure to hyperoxia. Both iNOS(+/+) and iNOS(-/-) mice were exposed to 100% O2 or room air for 55 h in specially designed environmental chambers and were housed in a temperature-controlled environmental facility, as previously described (50). The cages were flushed with either air or 100% O2 for 30 min, and then gas flow was maintained at 8 l/min, which resulted in nondetectable levels of carbon dioxide in the outflow gas. The O2 concentration in the chamber was monitored continuously with an O2 analyzer (Ohmeda 5120) and was maintained above 95% in the O2 group. Mice had access to food and water ad libitum throughout the exposure period.

Plasma osmolality. iNOS(+/+) and iNOS(-/-) mice were killed with intraperitoneal injections of ketamine (8.7 mg/100 g body wt; Phoenix Scientific, St. Joseph, MO) and xylazine (1.3 mg/100 g body wt; Vedco, St. Joseph, MO). The brachial artery and vein were severed, 1 ml of blood was collected in tubes containing 3% EDTA and centrifuged at 9,400 g for 10 min, plasma was collected, and plasma osmolality was measured using a vapor pressure osmometer (Vapro; Wescor, Logan, UT).

AFC measurement. iNOS(+/+) and iNOS(-/-) mice were anesthetized with an intraperitoneal injection of diazepam (5 mg/kg body wt; Abbott Laboratories, Chicago, IL) followed 10 min later by intraperitoneal ketamine (200 mg/kg; Phoenix Scientific) and were placed on a heating pad (Braintree, Cambridge, MA). Temperature was monitored continuously with a temperature probe placed under the skin and was maintained at 37°C using a heating pad and lamp. The trachea was exposed and cannulated with an 18-gauge intravenous catheter trimmed to ~0.5 in.; the catheter was connected to a mouse respirator (model 687; Harvard Apparatus, Holliston, MA) via a three-way valve. All mice were paralyzed with an intraperitoneal injection of pancuronium bromide (0.04 mg; Gensia Pharmaceuticals, Irvine, CA) and were ventilated with 100% O2 with a tidal volume of 0.2 ml (9-10 ml/kg body wt) and frequency of 160 breaths/min. Preliminary studies showed that mice instilled with fluid for measurement of AFC (see below) while ventilated in such a fashion maintained adequate oxygenation and acid-base balance during the time course of the experiment. Heart rate and rhythm were monitored continuously via a three-lead electrocardiogram (Physio-Control, Redmond, WA).

Once a stable level of anesthesia was obtained, judged by heart rate and lack of toe reflex, mice were positioned in the left decubitus position, and 0.3 ml of either NaCl or N-methyl-D-glucamine chloride (NMDG+Cl-; with or without amiloride and/or forskolin, depending on the experimental group) containing 5% fatty acid-free BSA (Sigma, St. Louis, MO) was instilled in the tracheal cannula over 30 s. We infused 0.1 ml of room air in the catheter to clear the dead space and position the fluid in the alveolar space. The osmolality of the instilled fluid was adjusted to that of the plasma for the particular group of mice by addition of the appropriate salt before instillation. All mice were ventilated for a 30-min period at which time the alveolar fluid was aspirated by applying gentle suction to the tracheal catheter with a 1-ml syringe. The volume of the recovered fluid was 0.05-0.1 ml. AFC, expressed as a percentage of total instilled volume, was calculated from the following relationship, as described previously (47, 49)
AFC<IT>=</IT>(1<IT>−</IT>C<SUB>i</SUB><IT>/</IT>C<SUB>30</SUB>)<IT>/</IT>0.95
where Ci and C30 are, respectively, the protein concentration of the instillate before instillation and of the alveolar sample at 30 min, each measured using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). At the end of some experiments, an arterial blood sample was drawn from the carotid artery using a 28-gauge heparanized insulin syringe for the measurement of arterial blood gases and pH using a system 1306 pH/blood gas analyzer (Instrumentation Laboratory).

AFC was measured in seven groups of iNOS(+/+) and iNOS(-/-) mice, five without and two with hyperoxic exposure. In addition to 5% BSA, solutions contained NaCl, NaCl with 1.5 mM amiloride, NMDG+Cl-, NaCl with 50 µM forskolin, and NaCl with 50 µM forskolin and 1.5 mM amiloride. None of the solutions contained glucose. The concentrations of amiloride in the instillate and in the alveolar fluid 30 min after instillation were measured spectrophotometrically at 361 nm (Em = 0.02 µM-1). The concentration of amiloride in the instillate resulting in the maximum inhibition of AFC was determined in preliminary studies.

Measurements of alveolar fluid volume and albumin efflux. Two critical assumptions in the measurement of AFC are that 1) the instilled protein is not diluted significantly by the presence of alveolar fluid and 2) there is no significant flux of albumin across the alveolar epithelium during the experimental period. To test the first assumption, the alveolar instillate was withdrawn 30 s after its instillation, and its protein concentration was measured. The quantity of alveolar fluid leaving the alveolar space within 30 s from its instillation will be minimal, so changes in albumin concentration reflect the amount of alveolar edema present. To test the second assumption, we instilled isosmotic NaCl containing 5% BSA and 1 µCi of 125I-labeled albumin (specific activity = 1.954 mCi/ml; ICN, Irvine, CA) into the alveolar spaces of mice from the various experimental groups. All other procedures were as described above. A blood sample was taken 30 min postinstillation, and its 125I radioactivity, along with that of a sample of the instillate, was measured using a gamma counter (Automatic Gamma System model 1285; Tm Analytic, Elk Grove Village, IL). The amount of 125I in the mouse blood as a fraction of 125I in the instillate was calculated from the following equation
(<SUP>125</SUP>I counts<IT>/</IT>ml of blood)<IT>×</IT>(weight of mouse)

<IT>×</IT>0.07<IT>/</IT>(total <SUP>125</SUP>I counts in the instillate)
where 0.07 is an estimate of blood volume. To determine the fraction of unbound 125I, we added TCA to some alveolar and blood samples to precipitate albumin, centrifuged them, and measured radioactivity in both the pellet and the supernatant. In all cases, supernatant (free 125I) radioactivity was <1% of its protein-associated value.

Lung wet-to-dry weight ratio measurement. Uninstilled mice were killed with an intraperitoneal injection of ketamine and xylazine (as above). The chest was opened, and the heart and lungs were removed en bloc. Nonpulmonary tissue was carefully dissected away, and the lungs were blotted, weighed, and placed in an oven at 55°C for 7 days. At that time, the lungs were weighed again, and the wet-to-dry weight ratio was calculated. In a separate group of mice, the lungs were homogenized, and hemoglobin concentration of the homogenate and a blood sample were measured using a total hemoglobin kit (Sigma Diagnostics). The ratio of the blood-free wet-to-dry lung was then calculated as previously described (31). Because of the very small size of the mouse lungs, hemoglobin concentrations in the lung homogenates were very small and barely above background levels. Thus measurements of the bloodless wet-to-dry lung weights were not considered reliable and are not reported here.

Quantitative histopathology. iNOS(+/+) and iNOS(-/-) mice exposed to 100% O2 for 55 h or normoxic conditions were killed by an intraperitoneal injection of ketamine-xylazine followed by severance of the brachial blood vessels (as above). The larynx, trachea, and thoracic organs were removed en bloc. The lungs were infused via the trachea to approximately normal distension with 10% formalin in 70% ethanol and then fixed in dorsal recumbency for 24 h in this solution. After fixation, each of the five lobes was trimmed along its airway and vascular arborization, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin for light microscopy. To confirm loss of cross striations in pulmonary veins, normally composed of cardiac muscle (39), some sections were stained by Mallory's phosphotungstic acid-hematoxylin method. All sections were coded randomly, and each lobe was scored subjectively (0-4) for severity of each of the following anatomic changes: 1) alveolar hemorrhage; 2) alveolar edema; 3) interstitial (peribronchial, peribronchiolar, and perivascular) edema; 4) interstitial inflammation; 5) pulmonary vein degeneration; and 6) degeneration and sloughing of bronchiolar epithelium. Scores for each lesion were weighted according to the percentage that each lobe contributes to total lung weight in arriving at a total lesion score for each set of lungs. For each of the six lesions, a "lesion index" (LI) was calculated by dividing the observed lesion score by the maximum score possible. Thus the maximum LI possible was 1.0. The "total lung lesion score" was calculated by adding all of the lesion indexes for a set of lungs.

Bronchoalveolar lavage. Mice were killed with an intraperitoneal injection of ketamine-xylazine, and a trimmed sterile 18-gauge intravenous catheter (Surflo; Terumo Medical, Elkton, MD) was inserted caudally into the lumen of the exposed trachea. The lungs were then lavaged in situ with three separate 1-ml washes of sterile saline. The bronchoalveolar lavage (BAL) fluid was centrifuged, and the cellular pellet was resuspended in DMEM containing 2% BSA and 2.5 g/l HEPES (pH 7.4). Total leukocyte count was determined using a hemocytometer. Protein concentrations of the lavage supernatants were measured using the BCA protein assay (Pierce). Samples to be used for immunohistochemistry were separated into aliquots in Lab-Tek sterile chamber slides (Nunc, Naperville, IL) at a concentration of 104 cells/well. Cells were incubated at 37° for 1 h, washed with PBS, fixed with 4% parformaldehyde for 10 min, and washed with PBS again. Slides were stored in 70% ethanol at -20°C until staining.

Immunocytochemistry for iNOS. Slides containing cells from BAL fluid from iNOS(+/+) and iNOS(-/-) mice were rinsed in PBS and permeabilized with 50 mM lysine-0.1% Triton X-100 for 15 min. Endogenous peroxidase activity was quenched by treatment with 0.3% hydrogen peroxide in cold methanol for 30 min followed by three washes with PBS. Nonspecific binding was blocked with 10% goat serum and 10% mouse serum in PBS for 2 h. The primary antibody, anti-mouse iNOS, NH2-terminus, rabbit polyclonal IgG (Upstate Biotechnology, Lake Placid, NY), or nonspecific IgG (both at 0.01 mg/ml in 10% goat serum and 2% BSA in PBS) was applied to the cells overnight. After being washed in 0.1% Triton in PBS, cells were incubated with the goat anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody from a DAKO EnVision system kit (DAKO, Carpintera, CA) for 30 min at 37°C. The cells were then rinsed in 50 mM Tris · HCl and developed in a filtered solution of diaminobenzidine from the same kit for 30 s. Slides were counterstained with methyl green (Fisher Scientific, Fair Lawn, NJ). For each slide, at least 300 cells were counted and graded as positive or negative based on the presence or absence of the brown reaction product in the cell cytoplasm.

Nasal potential difference measurement. Mice were anesthetized by an intraperitoneal injection of a mixture of ketamine (33 mg/100 g body wt; Fort Dodge Laboratories, Fort Dodge, IA), acepromazine (1.2 mg/100 g body wt; Vedco), and xylazine (6 mg/100 g body wt; Vedco). Mice were then placed on their backs, their tail was immersed in 0.9% NaCl, and a catheter was placed in their nasal passages, as previously described (13). The nasal passages were then perfused with lactate Ringer or lactate Ringer with 100 µM amiloride at a rate of 3 µl/min using a perfusion pump. The potential difference across the nares with the tail being the reference was measured with a voltmeter and recorded on a strip chart recorder at a speed of 30 cm/h. Solutions were changed only after the voltage had stabilized.

Statistics. All data were analyzed by ANOVA, using the Bonferroni method for multiple comparisons or Student's t-test when appropriate. All values given are means ± SD, and a P value <0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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AFC in normoxic mice. Arterial blood gases in anesthetized mice 30 min after fluid instillation are shown in Table 1. Both iNOS(+/+) and iNOS(-/-) mice had normal acid-base balance and arterial PO2 (PaO2) values >80 Torr, consistent with adequate tissue oxygenation and perfusion. The lower than expected PaO2 values for this level of inspired O2 are consistent with the presence of a right-to-left shunt resulting from the instillation of fluid in the alveolar space. iNOS(-/-) mice had significantly higher plasma osmolality compared with iNOS(+/+) mice [334 ± 5 vs. 321 ± 5 (SD) mosmol/kgH2O; n = 9; P < 0.001]. We thus adjusted the instillate osmolality to match the plasma osmolality for each group of mice.

                              
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Table 1.   Physiological measurements in anesthetized mice

Both iNOS(+/+) and iNOS(-/-) mice had high levels of AFC (33 ± 6 vs. 29 ± 7% of instilled fluid/30 min, respectively; mean ± SD, n = 10 each). Because the mean dry weights of the iNOS(+/+) and iNOS(-/-) lungs were 34.65 and 30.7 mg, respectively, and we instilled 0.3 ml of fluid in both groups of mice, we calculated AFC values of 5.7 and 5.6 ml · h-1 · g dry lung wt-1 for the iNOS(+/+) and iNOS(-/-) mice, respectively. Substitution of NaCl with equimolar concentrations of NMDG+Cl- in the instillate decreased AFC in the iNOS(+/+) mice to -9 ± 10% of instillate/30 min (mean ± SD; n = 7). Interestingly, when iNOS(-/-) mice were instilled with the NMDG+Cl- solution, the alveolar fluid protein concentration decreased significantly during the course of the experiment, resulting in a "negative" AFC [-24 ± 7% of instillate/30 min, mean ± SD; n = 7; P < 0.05 compared with the corresponding value in iNOS(+/+) mice]. Because the amount of 125I-albumin detected in the blood was extremely small, indicating no significant amount of albumin loss during the experimental period, and the AFC at 30 s was not different from zero, indicating no significant amount of alveolar edema in the lungs of these mice, this negative AFC resulted from net secretion of fluid in the alveolar space.

As shown in Fig. 1, addition of amiloride (1.5 mM) in the instillate containing NaCl resulted in a 61% decrease of AFC in the iNOS(+/+) mice but had no effect on AFC in the iNOS(-/-) mice. We chose this concentration of amiloride since previous studies had shown that instilled amiloride is cleared rapidly across the airspaces (37). Indeed, alveolar concentration of amiloride in the instillate of both iNOS(+/+) and iNOS(-/-) mice decreased from its initial value of 1.5 to 0.34 ± 0.05 (SD) mM (n = 10; P < 0.05) at the end of the experiment. At a lower concentration (1 mM), amiloride did not decrease AFC and could not be detected in the alveolar instillate at the end of the experimental period.


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Fig. 1.   Measurements of alveolar fluid clearance (AFC) in anesthetized, ventilated inducible nitric oxide synthase (iNOS)-deficient [iNOS(-/-)] and wild-type control [iNOS(+/+)] mice. AFC was calculated from changes in instilled BSA concentration during a 30-min period. Data are means ± SD (n >=  6 animals for each group). In all cases, the instillate contained 5% fat-free BSA in NaCl or N-methyl-D-glucamine chloride (NMDG+Cl-) as indicated. The osmolality was adjusted to that of the plasma for the appropriate group. Amiloride (amil; 1.5 mM) and/or forskolin (forsk; 50 µM) was also added to the instillate as indicated. *P < 0.05 compared with the corresponding control (Cont) iNOS(+/+) value. #P < 0.05 compared with the corresponding iNOS(+/+) value for the same experimental conditions.

To evaluate the effects of ouabain on AFC, three C57BL/6 iNOS(+/+) and three iNOS(-/-) mice were given a single intraperitoneal injection of 0.8 µg of ouabain. The mice were then anesthetized, and 30 min later, an isosmotic NaCl solution containing 5% albumin and 1.5 mM ouabain was instilled in their alveolar space, as described in MATERIALS AND METHODS. All mice developed atrial-ventricular heart block (as evidenced by continuous electrocardiogram recordings) 5 min after the intraperitoneal ouabain injection. AFC was measured 30 min after instillation when mice had been dead for at least 15 min. Results were as follows [mean ± SD; control (n = 6) vs. ouabain (n = 3)]: 1) iNOS(+/+): 32.1 ± 6.4 vs. 28.5 ± 7.0; 2) iNOS(-/-): 29.3 ± 7.3 vs. 23.7 ± 4.3. The ouabain values were not statistically significant from their corresponding controls.

Addition of 50 µM forskolin to the instillate increased AFC in the iNOS(+/+) mice from 33 ± 6 to 51 ± 4% of instillate/30 min (mean ± SD; n = 5; P < 0.05). This increase was the result of upregulation of amiloride-insensitive pathways, since amiloride decreased its value to only 36 ± 6% of instillate/30 min (mean ± SD; n = 6), which is significantly higher than the corresponding value in the presence of amiloride and absence of forskolin (13 ± 6, mean ± SD; n = 6, Fig. 1). In contrast, forskolin had no effect on AFC of iNOS(-/-) mice (Fig. 1).

Nasal potential difference measurement. iNOS(+/+) and iNOS(-/-) mice had similar baseline nasal potential difference (NPD) values (-8.3 ± 1.5 vs. -6.2 ± 2.7 mV; mean ± SD; n = 6; apical membrane compared with tail). Perfusion of the external nares of the iNOS(+/+) mice with lactate Ringer solution containing 100 µM amiloride resulted in a change in NPD of 3.8 ± 1.6 mV, consistent with depolarization of the apical membrane resulting from the blockade of Na+ entry pathways (Fig. 2A). On the other hand, no significant change in NPD was observed when the external nares of iNOS(-/-) mice were perfused with an amiloride-containing solution (change in NPD = 1 ± 1.4 mV; Fig. 2B). These findings are consistent with those shown in Fig. 1 and indicate the lack of amiloride-sensitive Na+ transport across both the alveolar and nasal epithelium of iNOS(-/-) mice.


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Fig. 2.   A: representative measurements of nasal potential difference (NPD) across the external nares of iNOS(+/+) and iNOS(-/-) mice. The nares were initially perfused with lactate Ringer. Once a stable value of the potential difference was obtained, the nares were perfused with lactate Ringer containing 100 µM amiloride. Typical records were repeated in 6 different iNOS(+/+) and iNOS(-/-) mice. B: amiloride-induced changes in NPD of iNOS(+/+) and iNOS(-/-) mice. Values are means ± SD; n = 6. **P < 0.01.

Exposure to 100% O2. None of the iNOS(+/+) or iNOS(-/-) mice died during exposure to 100% O2 for 55 h. Both groups of mice developed sublethal lung injury as shown by flaring of the nostrils and higher lung wet-to-dry weight ratios, total lung lesion scores, and BAL fluid protein concentrations compared with air-breathing controls (Table 2). However, iNOS(-/-) mice developed less lung injury as shown by a 15% increase of the lung wet-to-dry weight ratios compared with 33% in the iNOS(+/+) mice and a 160% increase in total lung lesion score compared with 400% for iNOS(+/+) mice. These data are consistent with higher endothelial injury in the iNOS(+/+) mice. Lungs from iNOS(+/+) and iNOS(-/-) mice exposed to either hyperoxic or normoxic conditions were evaluated by subjective quantification of the following six morphological lesions: 1) alveolar hemorrhage; 2) alveolar edema; 3) interstitial (peribronchial, peribronchiolar, and perivascular) edema; 4) interstitial inflammation; 5) pulmonary vein degeneration; and 6) sloughing of bronchiolar epithelium. No alveolar hemorrhage or edema was found in any of the mice. Mild interstitial edema and inflammation (lymphocytic and neutrophilic) were observed, but differences between groups were not statistically significant. In both iNOS(+/+) and iNOS(-/-) mice exposed to hyperoxia, there was hyalinization and patchy to diffuse loss of cross striations in cardiac muscle, and the severity of this lesion was significantly greater in iNOS(+/+) mice (LI = 0.51 ± 0.03) than in iNOS(-/-) mice (LI = 0.26 ± 0.13; Table 2). Pulmonary veins were histologically normal in all mice of both strains exposed to normoxic conditions. In addition, epithelium in the bronchioles of iNOS(+/+) and iNOS(-/-) mice exposed to hyperoxia had diffuse, moderate swelling and increased eosinophilia of apical cytoplasm, with sloughing of nonciliated and ciliated epithelial cells in their lumens. Sloughed cells were rounded, pyknotic, and frequently undergoing karyolysis. In some bronchioles, cell losses resulted in patchy ulcerations. Severity of these changes was significantly greater in iNOS(-/-) mice (LI = 0.56 ± 0.04) compared with iNOS(+/+) mice (LI = 0.22 ± 0.2). Both strains of mice had morphologically normal bronchioles after exposure to normoxic conditions.

                              
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Table 2.   Effects of breathing 100% O2 on iNOS(+/+) and iNOS(-/-) mice

iNOS immunocytochemistry. Cells (300) isolated from the BAL fluid of each group of mice [iNOS(+/+) or iNOS(-/-) breathing air or 100% O2] were counted after immunostaining with a polyclonal anti-mouse iNOS antibody. In all cases, >95% of these cells were alveolar macrophages. Approximately 2 and 92% of cells isolated from the BAL fluid of air- and O2-breathing iNOS(+/+) mice stained positive for iNOS, respectively. Typical fields are shown in Fig. 3. In contrast, none of the cells from iNOS(-/-) mice stained positive for iNOS.


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Fig. 3.   iNOS immunocytochemistry in cells from bronchoalveolar lavage (BAL). Cells were isolated from the BAL fluid of iNOS(+/+) and iNOS(-/-) mice breathing either 100% O2 for 55 h or room air and were immunostained with a polyclonal antibody to iNOS followed by a secondary antibody (goat anti-rabbit IgG conjugated to horseradish peroxidase). A: cells from control (air-breathing) iNOS(+/+) mouse immunostained with the iNOS antibody. B: cells from an iNOS(+/+) mouse after breathing 100% O2 for 55 h immunostained with the iNOS antibody. C: cells from an iNOS(-/-) mouse after breathing 100% O2 immunostained with the iNOS antibody. D: cells from iNOS(+/+) mouse after breathing 100% O2 for 55 h stained with an equivalent amount of nonspecific IgG instead of the primary antibody. Representative fields are shown; data were reproduced 3 times using cells from 3 different mice in each category.

AFC in hyperoxic mice. Exposure of iNOS(+/+) or iNOS(-/-) mice to 100% O2 for 55 h did not alter their basal AFC levels (Fig. 4). However, exposure of iNOS(+/+) mice to hyperoxia decreased their amiloride-sensitive portion of AFC from 61 to 40% of total (Fig. 4). Amiloride (1.5 mM) did not alter AFC of iNOS(-/-) mice exposed to hyperoxia, similar to what was observed in normoxia. Hyperoxic exposure did not increase the 30-s AFC value or the amount of 125I-albumin in the blood (Table 2).


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Fig. 4.   Effects of breathing 100% O2 for 55 h on AFC of iNOS(+/+) and iNOS(-/-) mice. Mice were exposed to either room air or 100% O2 for 55 h in environmental chambers as described in text. They were then removed from the chambers, anesthetized, and ventilated with 100% O2, and AFC (in the presence and absence of 1.5 amiloride) was measured as described in the text. Values are means ± SD (n >=  6 for each group). *P < 0.05 for amiloride vs. the corresponding control value for the indicated group of mice. #P < 0.05 between the 2 amiloride values.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The availability of transgenic mice and the considerable interest in understanding basic mechanisms responsible for the regulation of fluid transport across the alveolar space in vivo have necessitated the development of new techniques to measure AFC in mice under physiological conditions. The studies of Garat et al. (12) and Icard and Saumon (18) were the first to report successful measurements of amiloride-sensitive alveolar fluid reabsorption across the lungs of c57 (presumably C57BL/6) mice in situ and isolated perfused lungs of CD-1 mice, respectively. However, Fukuda et al. (11) reported recently that lungs of mice in situ developed a significant amount of interstitial edema, most likely because of the lack of pulmonary lymph and blood flow, which inhibited AFC by ~30%. In their studies, Fukuda et al. measured AFC in anesthetized, ventilated mice but did not report values for arterial blood gases during the experimental period. We ventilated our mice with a tidal volume of 9-10 ml/kg body wt, similar to what they reported. However, to maintain adequate oxygenation and acid-base balance, we had to ventilate the mice using a much higher respiratory frequency than Fukuda et al. reported (160 vs. 90 breaths/min). Furthermore, we carefully adjusted the osmolality of the instillate to that of the plasma for each strain of mice [321 ± 5 and 334 ± 5 mosmol/kgH2O in the iNOS(+/+) and iNOS(-/-) mice, respectively], since the presence of an osmotic gradient across the alveolar epithelium may complicate the interpretation of AFC.

Comparison of our results with previous studies is complicated not only by the fact that our measurements were done under normal physiological conditions but also by differences in genotype. For example, although our basal values of AFC in iNOS(+/+) mice are similar to those reported by Fukuda et al. (11), Ma et al. (30), and Icard and Saumon (18; ~30% of instilled fluid/30 min corresponding to ~6 ml · h-1 · g dry lung wt-1), the fraction of AFC blocked by amiloride was very different (~60% of AFC in our case vs. >90% in the above-mentioned reports). This was most likely because these investigators used CD-1 mice while we used C57BL/6 mice. Indeed, in a previous study, Garat et al. (12) reported a 40% inhibition of AFC across the lungs by amiloride using the in situ model on C57BL/6 mice. These findings underscore the importance of using properly matched genetic controls in transgenic mice experiments.

Our studies show that both iNOS(+/+) and iNOS(-/-) mice have similar levels of AFC, which are totally dependent on the presence of active Na+ reabsorption, as shown by the lack of AFC when Na+ was replaced by NMDG+Cl-, a large positive ion with very small permeability across ATII cell Na+ channels (32). However, AFC in the iNOS(-/-) mice was totally insensitive to amiloride. This finding was consistent with the lack of amiloride sensitivity of the potential difference across the external nares. Amiloride-insensitive fluid clearance is very important in that it makes up ~50% of fluid clearance in most animals and in humans (2, 42). This study shows that these pathways can be increased, which could have clinical applications in the future.

Our NPD studies in iNOS(-/-) mice yielded comparable results to those performed by Elmer et al. (7), who found that only 25% of the baseline NPD was eliminated by amiloride compared with our 16%. They ascribed their finding to the background of the mice.

The lack of amiloride-sensitive fluid clearance in the iNOS(-/-) mice was unexpected and seems paradoxical considering that NO has been shown to downregulate amiloride-sensitive transport across alveolar epithelial cells via a cGMP-dependent mechanism (21, 26). These results imply that there is an optimum level of NO for the amiloride-sensitive Na+ channel (ENaC) or at least for its amiloride sensitivity. There are several possibilities that may account for the lack of amiloride-sensitive fluid clearance in iNOS(-/-) mice. The ENaC is a heteromultimeric complex of at least three distinct but homologous subunits, alpha -, beta - and gamma -ENaC, which were first cloned from the colon of salt-deprived rats and human lung tissue (3, 28, 48). Experiments with point mutations suggested that all three subunits are involved in the channel pore formation (45). Expression of alpha -rat (r)ENaC mRNA in adult rat ATII cells was demonstrated by Northern blot analysis (50), PCR (9), and in situ hybridization (9, 50). beta - and gamma -rENaC mRNAs were also detected in large and small airways, but they were less abundant in ATII cells compared with alpha -rENaC (9). Our previous biophysical and biochemical studies in A549 cells, an alveolar epithelial line, showed that alterations in the stoichiometry of the alpha -, beta -, and gamma -subunits of ENaC altered both the conductance of Na+ single channels and their response to amiloride. Specifically, incubation of A549 cells with dexamethasone increased expression of beta - and gamma -human (h)ENaC protein levels and decreased the amiloride half-maximal inhibitory concentration (K0.5) for whole cell currents from 833 ± 69 to 22 ± 5.4 nM (mean ± SE; P < 0.01; see Ref. 27). An increase in amiloride K0.5 for epithelial channels of ATII cells in iNOS(-/-) mice may manifest itself as a lack of response to amiloride.

Admittedly, the concentration of instilled amiloride was quite high (1.5 mM). However, measurements of the alveolar concentration of amiloride at the end of the experimental period were considerably and equally lower (~300 µM) in both types of mice, and its concentration in the epithelial lining fluid was most likely much lower because of its binding to albumin and nonuniform distribution in the alveolar epithelial lining fluid. This finding is consistent with the results of numerous previous studies showing that, in contrast to its very low inhibition constant (Ki) in vitro (20-30 nM), much larger concentrations (0.8-1.5 mM) are needed in the instillate to decrease Na+ transport in vivo (37). In addition, forskolin, an agent that increases intracellular cAMP levels, upregulated AFC in the iNOS(+/+) mice by increasing amiloride-insensitive fluid clearance but failed to increase AFC in the iNOS(-/-) mice. Previous studies in a variety of species have shown that intratracheal instillation of agents that increase cAMP upregulate either the amiloride-sensitive (30) or the amiloride-insensitive fraction of AFC (23). Patch-clamp studies have shown that an increase in cAMP levels of ATII cells results in significant upregulation of both the open time and open probability of amiloride-sensitive single channels (51). This finding argues against the possibility of inactive channels being present in the ATII cells of iNOS(-/-) mice.

Another possibility is that Na+ transport across the alveolar airway epithelium of iNOS(-/-) mice occurs through pathways other than ENaC. These include the Na+-glucose cotransporter SGLT1 (18), the Na+-H+ antiport (29), and cyclic nucleotide-gated cation channels, especially CNG1 (24, 46). SGLT1 has been shown to be present in mouse lung (18), but transport via this channel seems unlikely because the instillate in our experiments did not contain glucose. CNG1 has been shown to be present in rat lung, to conduct amiloride-insensitive, cGMP-stimulated Na+ current across cultured tracheal cells (46), and to mediate a substantial portion of fluid clearance in sheep (24).

Finally, the fact that neither amiloride nor ouabain decreased AFC across the alveolar epithelium of iNOS(-/-) mice may suggest that Na+ transport occurred through paracellular pathways. We believe that this is very unlikely because 1) since the instillate was isosmotic with the plasma, there was no driving force for the egress of either Na+ or fluid across the alveolar epithelium, and 2) ouabain also failed to decrease AFC in the iNOS(+/+) mice in which 60% of AFC was inhibited by amiloride. Addition of 0.1-1 mM ouabain to the instillate and to the perfusion solution of ex vivo animal or human lung preparations inhibited AFC from 50 to 100% (18, 30, 42-44). The only study evaluating the effects of ouabain in vivo was performed by Jayr et al. (23). These authors measured AFC in rats and reported that addition of 1 mM ouabain to the instillate and simultaneous intravenous infusion of 0.004 mg of ouabain decreased AFC from 33 ± 9 to 23 ± 4 (net change in AFC = 30%). As mentioned, our data indicate that intraperitoneal and intratracheal instillation of ouabain resulted in progressive atrioventricular block and death of the mice. Because of the severe hemodynamic alterations, which most likely effected the measurements of AFC, and the uncertainty as to whether we achieved a sufficiently high ouabain concentration on the basolateral membranes of ATII cells, these studies were not pursued further.

Our previous studies and those of others showed that reactive oxygen nitrogen intermediates, formed by the reaction of NO with superoxide and thiols, decrease short-circuit current because of Na+ across cultured monolayers of ATII cells by damaging both apical and basolateral Na+-transporting pathways (14), decrease amiloride-sensitive whole cell currents across Xenopus oocytes injected with alpha -, beta -, and gamma -ENaC (6), and inhibit both whole and single channel currents across A549 (27), ATII (21), and fetal distal epithelial (4) cells. In addition, perfusion of the external nasal passages with an iNOS inhibitor resulted in upregulation of amiloride-sensitive Na+ transport, suggesting that tonic release of NO congeners by iNOS downregulated amiloride-sensitive transport across airway epithelial cells (7).

To gain additional insight into the role of endogenous NO in the regulation of Na+ transport across the alveolar epithelium in vivo, we exposed both iNOS(+/+) and iNOS(-/-) mice to a sublethal level of hyperoxia. Exposure to hyperoxia resulted in a significant upregulation of iNOS in inflammatory cells and damaged the alveolar epithelium as shown by the large increase in the wet-to-dry weight ratio and alveolar albumin concentration in both iNOS(+/+) and iNOS(-/-) mice. However, the degree of lung damage in iNOS(-/-) mice was lower than in iNOS(+/+) mice as shown by lower increases in the lung wet-to-dry weight ratio and overall lung index score. We found no change in basal AFC with 100% O2 for 55 h, but the amiloride-sensitive portion of fluid clearance decreased from 61% of total clearance with normoxia to 40% with hyperoxia in iNOS(+/+) mice. This was a result of upregulation of the amiloride-insensitive component of AFC by NO. Instillation of DETANONOate, an NO donor, in the alveolar spaces of rabbits also resulted in a decrease of the amiloride-sensitive portion of the AFC without deceasing its basal value (36). The mechanism responsible for this effect has not been elucidated.

In these studies, hyperoxia resulted in significant injury to cardiac muscle in pulmonary veins and to epithelium of bronchioles in both iNOS(+/+) and iNOS(-/-) mice. These changes were clearly degenerative in character and thus are most likely indicative of toxic effects of O2. It is not entirely clear whether these changes had any effect on ion transport, since ATII cells are generally accepted as mainly responsible for ion transport in the lungs. Although cardiac muscle does not occur in pulmonary veins of humans, as it does in mice, and humans have a much different population of cells (far fewer Clara cells) in their bronchioles than mice, our findings raise the possibility of toxic effects of hyperoxia on cardiovascular and bronchiolar tissues of patients.

In summary, our results indicate that iNOS(-/-) mice have normal levels of Na+-dependent fluid transport across their alveolar and airway epithelia in vivo. However, these mice either lack amiloride-sensitive pathways or the amiloride IC50 of their apical transporters is significantly higher than the corresponding value in their wild-type controls. Thus NO produced by iNOS may be important in the regulation of amiloride-sensitive Na+ pathways across these epithelia.


    ACKNOWLEDGEMENTS

We thank Judy Hickman-Davis, Ian Davis, Glenda Davis, Tanta Miles, Mark Philips, and James Fortenberry for technical support and many useful discussions.


    FOOTNOTES

This study was supported by National Institutes of Health Grants HL-51173, HL-31197, and P30-DK-54781 and by funds from the Veterans Affairs Research Service.

Address for reprint requests and other correspondence: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 19th St. S., THT 940, Birmingham, AL 35233 (E-mail: Sadis.Matalon{at}ccc.uab.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 31 January 2001; accepted in final form 17 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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