Inhibition of beta -adrenergic-dependent alveolar epithelial clearance by oxidant mechanisms after hemorrhagic shock

K. Modelska, M. A. Matthay, L. A. S. Brown, E. Deutch, L. N. Lu, and J. F. Pittet

Departments of Anesthesia and Medicine and Cardiovascular Research Institute, University of California, San Francisco, California 94143; and Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Endogenous release of catecholamines is an important mechanism that can prevent alveolar flooding after brief but severe hemorrhagic shock. The objective of this study was to determine whether this catecholamine-dependent mechanism upregulates alveolar liquid clearance after prolonged hemorrhagic shock. Rats were hemorrhaged to a mean arterial pressure of 30-35 mmHg for 60 min and then resuscitated with a 4% albumin solution. Alveolar liquid clearance was measured 5 h later as the concentration of protein in the distal air spaces over 1 h after instillation of a 5% albumin solution into one lung. There was no upregulation of alveolar liquid clearance after prolonged hemorrhagic shock and fluid resuscitation despite a significant increase in plasma epinephrine levels. The intravenous or intra-alveolar administration of exogenous catecholamines did not upregulate alveolar liquid clearance. In contrast, catecholamine-mediated upregulation of alveolar liquid clearance was restored either by depletion of neutrophils with vinblastine, by the normalization of the concentration of reduced glutathione in the alveolar epithelial lining fluid by N-acetylcysteine, or by the inhibition of the conversion from xanthine dehydrogenase to xanthine oxidase. These experiments provide the first in vivo evidence that a neutrophil-dependent oxidant injury to the alveolar epithelium prevents the upregulation of alveolar fluid clearance by catecholamines in the absence of a major alteration in paracellular permeability to protein after prolonged hemorrhagic shock.

alveolar epithelium; beta -adrenergic receptor; alveolar liquid clearance; neutrophil; oxidative injury


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TRAUMA AND HEMORRHAGIC SHOCK are two of the most common causes of acute lung injury in patients (26). Even though the mechanisms of lung endothelial injury after hemorrhagic shock have been studied by several investigators (19, 25, 36, 46, 58), the critical importance of the alveolar epithelial barrier in the pathogenesis and recovery from acute lung injury secondary to hemorrhagic shock has not been well examined. In addition, in patients with acute lung injury who develop pulmonary edema, the preservation of the capacity of the alveolar epithelium to actively remove fluid from the air spaces is crucial for their survival (34).

The results from earlier experimental studies from our laboratory (37, 45) indicate that the endogenous release of catecholamines is a major mechanism that prevents alveolar flooding after the onset of septic or short-term hemorrhagic shock by upregulating vectorial, sodium-dependent fluid transport across the alveolar epithelium. However, the impact of this protective mechanism on lung fluid balance and alveolar fluid transport several hours after fluid resuscitation from prolonged hemorrhagic shock has not been studied.

Therefore, the first objective of these studies was to determine whether the endogenous release of catecholamines can upregulate alveolar fluid clearance after prolonged hemorrhagic shock and fluid resuscitation. Because there was no upregulation of alveolar fluid clearance 6 h after prolonged hemorrhagic shock despite a 20-fold increase in plasma levels of epinephrine, the second objective was to maximize the response of beta -adrenergic receptors by the administration of exogenous beta -adrenergic agonists in hemorrhaged and fluid-resuscitated rats. Because exogenous beta -adrenergic agonists did not stimulate alveolar fluid clearance, the third objective was to test the hypothesis that a neutrophil-dependent injury to the alveolar epithelium might account for the absence of upregulation of alveolar fluid clearance after prolonged hemorrhagic shock. Because neutrophil depletion with vinblastine before hemorrhagic shock was associated with restoration of the response of the alveolar epithelium to beta -adrenergic agonists, the fourth objective was to test the hypothesis that oxidant-mediated injury may account for the inability of the alveolar epithelium to respond to beta -adrenergic-agonist stimulation after prolonged hemorrhagic shock and fluid resuscitation by two different approaches. First, N-acetylcysteine (NAC), a glutathione precursor and an oxygen radical scavenger, restored the capacity of the alveolar epithelium to respond to beta -adrenergic agonists by upregulating alveolar fluid clearance and normalized the concentration of glutathione in the alveolar epithelial lining fluid. Second, the inhibition of the conversion from xanthine dehydrogenase to xanthine oxidase (XO) by a tungsten-enriched, molybdenum-deficient diet or an allopurinol-supplemented diet restored the beta -adrenergic-mediated upregulation of alveolar fluid clearance in hemorrhaged rats. Therefore, taken together, these data provide the first in vivo evidence of an oxidant-mediated neutrophil-dependent decrease in the fluid transport capacity of the alveolar epithelium in the absence of a major alteration in paracellular permeability to protein after prolonged hemorrhagic shock.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The protocol for these studies was approved by the University of California, San Francisco Animal Research Committee.

Lung Barrier Function Studies

Surgical preparation and ventilation. Male Sprague-Dawley rats (n = 102) weighing 300-350 g were anesthetized with pentobarbital sodium (60 mg/kg ip), and anesthesia was maintained with 30 mg/kg of pentobarbital sodium every 2 h. An endotracheal tube (PE-220) was inserted through a tracheotomy. Pancuronium bromide (0.3 mg · kg-1 · h-1 iv) was given for neuromuscular blockade. Catheters (PE-50) were inserted into both carotid arteries to monitor systemic arterial pressure, obtain blood samples, and withdraw blood for the induction of prolonged hemorrhagic shock. A catheter was also inserted into the jugular vein to monitor central venous pressure. The rats were maintained in the left lateral decubitus position during the experiments and were ventilated with a constant-volume pump (Harvard, Millis, MA) with an inspired oxygen fraction of 1.0 and peak airway pressure of 8-12 cmH2O, supplemented with a positive end-expiratory pressure of 3 cmH2O. The respiratory rate was adjusted to maintain arterial PCO2 between 35 and 40 mmHg during the baseline period.

Preparation of instillate. A 5% bovine albumin solution was prepared with Ringer lactate and adjusted with NaCl to be isosmolar with the rat's circulating plasma as previously published (37). Anhydrous Evan's blue dye (0.5 mg) was added to the albumin solution to confirm the location of the instillate at the end of the study, and 1 µCi of 125I-labeled human serum albumin (Frosst Laboratories) was also added to the albumin solution. 125I-albumin served as the alveolar protein tracer in all experiments. A sample of the instilled solution was saved for total protein measurement, radioactivity counts, and water-to-dry weight ratio measurements so that the dry weight of the protein solution could be subtracted from the final lung water calculation. In addition, in some studies, epinephrine, salmeterol, or propranolol was added to the instillate.

General protocol. In all experiments, after surgery, heart rate and systemic blood pressure were allowed to stabilize for 60 min (Fig. 1A). The rat was placed in the left lateral decubitus position to facilitate liquid deposition into the left lung 300 min after the onset of hemorrhagic shock. Hemorrhagic shock was induced by withdrawing blood from the carotid artery to maintain a mean systemic arterial pressure of 30-35 mmHg for 60 min. This corresponded to the removal of 9-12 ml of blood. After 1 h of hemorrhagic shock, the rats were resuscitated with an intravascular 4% albumin solution in 0.9% NaCl over 30 min to maintain a central venous pressure < 8 mmHg as done before by Modelska et al. (37). The volume of 4% albumin solution administered was twice the amount of blood withdrawn.



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Fig. 1.   A and B: general experimental protocols (see METHODS for further explanation).

A vascular tracer, 1 µCi of 131I-labeled human albumin, was injected into the blood 270 min after the onset of hemorrhagic shock to calculate the flux of plasma protein into the lung interstitium as previously published (37). An alveolar tracer, 1 µCi of 125I-albumin in 3 ml/kg of the 5% bovine albumin solution, was instilled into the left lower lobe 30 min later to calculate the flux of protein from the air spaces into the circulating plasma. This tracer was instilled with a 1-ml syringe and a polypropylene tube (0.5-mm ID) over 20 min.

At the end of the experiment (1 h after the beginning of the alveolar instillation), the abdomen was opened, and the rats were exsanguinated by transecting the abdominal aorta. Urine was obtained for radioactivity counts. The lungs were removed through a median sternotomy. An alveolar fluid sample (0.1-0.2 ml) from the distal air spaces was obtained by gently passing the sampling catheter (PE-50 catheter, 0.5-mm ID) into a wedged position in the instilled area of the left lower lobe. After centrifugation, the total protein concentration and radioactivity of the liquid samples were measured. The right and left lungs were homogenized separately for water-to-dry weight ratio measurements and radioactivity counts.

In experiments designed to achieve XO depletion (Fig. 1B), inhibition of conversion from xanthine dehydrogenase to XO was achieved by either a tungsten-enriched, molybdenum-deficient diet (0.7 g sodium tungstate/kg chow; ICN Biochemicals) given for 4 wk or an allopurinol-supplemented diet (50 mg/kg chow; ICN Biochemicals) given for 1 wk before hemorrhagic shock (11). Control rats that were fed a regular protein diet were not XO depleted.

Plasma levels of epinephrine were measured at three time points: just before the onset of hemorrhagic shock, at the end of the ischemic phase of hemorrhagic shock, and at the end of each experiment.

Specific Protocols

Group 1: Effect of prolonged hemorrhagic shock and fluid resuscitation on alveolar liquid clearance. The rats were hemorrhaged and resuscitated to determine the effect of prolonged hemorrhagic shock and fluid resuscitation on fluid transport across the lung epithelium. Five hours after the onset of hemorrhagic shock, 3 ml/kg of the 5% bovine albumin solution with 1 µCi of 125I-albumin were instilled into the left lung (n = 6 rats). Control studies included rats (n = 10) that underwent the same surgical preparation and were studied for the same period of time but were neither hemorrhaged nor fluid resuscitated. Additional experiments that included control and hemorrhaged rats (n = 6) were done to measure myeloperoxidase activity in the lung as a quantitative assessment of neutrophil infiltration into the lung 6 h after the onset of hemorrhagic shock.

Group 2: Stimulation with exogenous beta -adrenergic agonists after prolonged hemorrhagic shock. GROUP 2A: ADMINISTRATION OF EPINEPHRINE. Because there was no upregulation of alveolar fluid clearance after prolonged hemorrhagic shock, epinephrine was either given intravenously at a concentration of 0.08 mg · kg-1 · min-1 (n = 5 rats) or added to the protein solution instilled into the distal air spaces at a concentration of 10-5 M (n = 4 rats). This concentration of intravenous epinephrine has previously been shown in pilot experiments to be sufficient to upregulate alveolar liquid clearance. Control studies included rats that were not hemorrhaged nor fluid resuscitated but either received an intravenous infusion of epinephrine for 6 h at a concentration of 0.08 mg · kg-1 · min-1 (n = 4 rats) or had their distal air spaces instilled with an albumin solution containing 10-5 M epinephrine for the last hour of the experiment (n = 3 rats).

GROUP 2B: INSTILLATION OF SALMETEROL. Because there was no upregulation of alveolar liquid clearance in hemorrhaged rats treated with exogenous epinephrine given intravenously or into the distal air spaces, the next series of experiments included rats (n = 4) that had their air spaces instilled with an albumin solution containing salmeterol (10-5 M), a specific long-acting beta -adrenergic agonist. Control studies included rats (n = 3) that were not hemorrhaged nor fluid resuscitated but were instilled with an albumin solution containing salmeterol (10-5 M).

Group 3: Effect of air space instillation of a beta -adrenergic antagonist after prolonged hemorrhagic shock. To determine whether prolonged hemorrhagic shock and fluid resuscitation affect basal and/or stimulated alveolar liquid clearance, a beta -adrenergic antagonist, propranolol (10-4 M), was added to the solution instilled into the distal air spaces of the lung of hemorrhaged rats. In addition, rats (n = 3) were given epinephrine intravenously at a concentration of 0.08 mg · kg-1 · min-1. Control studies included rats (n = 3) that were not hemorrhaged nor fluid resuscitated but were given epinephrine intravenously (0.08 mg · kg-1 · min-1) and were instilled with an albumin solution containing propranolol (10-4 M). Pittet et al. (44) have previously reported that propranolol (10-4 M) does not affect the rate of nonstimulated basal alveolar liquid clearance.

Group 4: Neutrophil depletion with vinblastine. To determine whether a neutrophil-dependent injury to the alveolar epithelium might account for the absence of upregulation of alveolar fluid clearance after prolonged hemorrhagic shock, experiments included rats (n = 3) that were pretreated with vinblastine (0.75 mg/kg) intravenously 4 days before the onset of hemorrhagic shock to deplete circulating neutrophils as done before by Folkesson et al. (18). Epinephrine (10-5 M) was added to the protein solution instilled into the distal air spaces of the lung. The first series of control studies included rats (n = 4) that were pretreated with vinblastine and had their distal air spaces instilled with epinephrine solution (10-5 M) but did not undergo hemorrhagic shock and fluid resuscitation. To exclude the possibility that the effect of vinblastine could be explained by nonspecific cytopathologic effects, a second series of control studies included rats (n = 4) that were pretreated with vinblastine 1 day before the onset of hemorrhagic shock.

Group 5: Pretreatment with an oxygen radical scavenger, NAC. To determine whether the inability of beta -adrenergic-receptor agonists to upregulate fluid transport across the alveolar epithelium after prolonged hemorrhagic shock and fluid resuscitation was mediated through oxidant-mediated mechanisms, this series of experiments included rats (n = 4) that were pretreated with an oxygen radical scavenger, NAC (150 mg/kg; Sigma), given intravenously before the onset of hemorrhagic shock. Epinephrine (10-5 M) was added to the protein solution instilled into the distal air spaces of the lung. Control studies included rats (n = 4) that were pretreated with NAC (150 mg/kg) and had their air spaces instilled with epinephrine solution (10-5 M) but did not undergo hemorrhagic shock and fluid resuscitation. In pilot experiments, we found that pretreatment with NAC (150 mg/kg) did not affect the rate of alveolar liquid clearance in control rats (n = 4).

Group 6: Inhibition of the conversion from xanthine dehydrogenase to XO. To determine whether depletion of XO, an enzyme that has been associated with the production of oxygen radicals, would restore the ability of the alveolar epithelium to respond to beta -adrenergic agonists by upregulating alveolar liquid clearance, the rats were fed either a tungsten-enriched, molybdenum-deficient diet or an allopurinol-supplemented diet.

GROUP 6A: EFFECT OF TUNGSTEN-ENRICHED, MOLYBDENUM-DEFICIENT DIET ON ALVEOLAR LIQUID CLEARANCE IN RATS THAT UNDERWENT PROLONGED HEMORRHAGIC SHOCK AND FLUID RESUSCITATION. Rats (n = 6) were fed a tungsten-enriched, molybdenum-deficient diet (0.7 g sodium tungstate/kg chow) for 3 wk. Then they were hemorrhaged and fluid resuscitated. Epinephrine (10-5 M) was added to the protein solution instilled into the distal air spaces of the lung.

Control studies included rats (n = 4) that were fed a tungsten-enriched, molybdenum-deficient diet and had their air spaces instilled with epinephrine solution (10-5 M) but did not undergo hemorrhagic shock and fluid resuscitation. In pilot experiments, we found that a tungsten-enriched, molybdenum-deficient diet did not affect the rate of alveolar liquid clearance in control rats (n = 4).

GROUP 6B: EFFECT OF ALLOPURINOL-SUPPLEMENTED DIET ON ALVEOLAR LIQUID CLEARANCE IN RATS THAT UNDERWENT PROLONGED HEMORRHAGIC SHOCK AND FLUID RESUSCITATION. Rats (n = 6) were fed an allopurinol-supplemented diet (50 mg/kg chow) for 1 wk. Then they were hemorrhaged and fluid resuscitated. Epinephrine (10-5 M) was added to the protein solution instilled into the distal air spaces of the lung.

Control studies included rats (n = 4) that were fed an allopurinol-supplemented diet and had their air spaces instilled with epinephrine solution (10-5 M) but did not undergo hemorrhagic shock and fluid resuscitation. In pilot experiments, we found that an allopurinol-supplemented diet did not affect the rate of alveolar liquid clearance in control rats (n = 4).

Bronchoalveolar Lavage Studies

To determine whether oxidative injury to the alveolar epithelium secondary to prolonged hemorrhagic shock was associated with a decrease in the concentration of reduced glutathione (GSH) in the alveolar epithelial lining fluid, a first series of experiments included rats (n = 4) that were hemorrhaged and fluid resuscitated as described in General protocol. Then the rats were exsanguinated, and their lungs were lavaged through a tracheotomy with HEPES (10.3 mM)-phosphate buffer (2.59 mM), pH 7.4, containing NaCl (133 mM), KCl (5.2 mM), CaCl2 (1.89 mM), and MgCl2 (1.29 mM). The lungs were then inflated to total lung capacity (10 ml) and lavaged once. The lavage samples were then centrifuged at 800 g for 10 min at 4°C to remove cells and quickly frozen at -70°C.

A second series of experiments included rats (n = 4) that were pretreated with NAC before they underwent prolonged hemorrhagic shock and fluid resuscitation as described in General protocol. At the end of the experiments, the lungs were then inflated to total lung capacity (10 ml) and lavaged once.

A third series of experiments included control rats (n = 4) that were neither hemorrhaged nor fluid resuscitated. At the end of experiments, the lungs were then inflated to total lung capacity (10 ml) and lavaged once.

In each group, GSH and oxidized glutathione (GSSG) levels were measured in the alveolar epithelial lining fluid.

Measurements

Hemodynamics, pulmonary gas exchange, and protein concentration. Systemic arterial, central venous, and airway pressures were continuously measured. Arterial blood gases were measured at 1-h intervals. Samples from the instilled protein solution, the final distal air space fluid, and the initial and final blood were collected to measure total protein concentration with an automated analyzer (AA2 Technicon, Tarrytown, NY).

Albumin flux across endothelial and epithelial barriers. Two different methods were used to measure the flux of albumin across the lung endothelial and epithelial barriers as done before by our laboratory (37, 45). The first method measures residual 125I-albumin (the air space protein tracer) in the lungs as well as the accumulation of 125I-albumin in plasma. The second method measures 131I-albumin (the vascular protein tracer) in the extravascular space of the lungs.

The total quantity of 125I-albumin (the air space protein tracer) instilled into the lung was determined by measuring duplicate samples of the instilled solution for total radioactivity counts (in counts · min-1 · g-1) and multiplying this value by the total volume instilled into the lung. To calculate the residual 125I-albumin in the lungs at the end of the study, the average radioactivity counts of two 0.5-g samples obtained from the lung homogenate was multiplied by the total weight of the lung homogenate. The 125I-albumin count in the lung homogenate was added to the recovered count in the final aspirated distal air space fluid to calculate the quantity of instilled 125I-albumin that remained in the lungs at the end of the study. 125I-albumin in the circulating plasma was measured from a sample of plasma obtained at the end of the experiment. The plasma fraction was accounted for by multiplying the counts per minute per gram times the plasma volume [body weight × 0.07(1 - hematocrit)] (37, 45).

The second method requires measurement of the vascular protein tracer 131I-albumin in the extravascular space of the lungs. We estimated the quantity of plasma that entered the instilled lungs by measuring the accumulation of the vascular protein tracer 131I-albumin into the extravascular space of the instilled lung using the equation of plasma equivalents previously described (37, 45). The extravascular lung plasma equivalents were determined by the total count of 131I-albumin in the lung, subtracting the fraction in the blood in the lung as measured with the gravimetric method, and then dividing by the mean count in the circulating plasma over the duration of the experiment as previously done (37, 45).

Alveolar liquid clearance. Changes in the concentration of the nonlabeled bovine albumin and the instilled 125I-albumin over the study period (1 h) were used to measure the liquid clearance from the distal air spaces as done before by our laboratory (37, 45). There is a good correlation between the changes in the concentration of instilled nonlabeled bovine albumin and those of 125I-albumin. Because some reabsorption may have occurred across distal bronchial epithelium, the term alveolar does not imply that all fluid reabsorption occurred at the alveolar level.

Tracer binding measurement. To determine 125I binding to albumin, trichloroacetic acid (20%) was added to all tubes, which were then centrifuged to obtain the supernatant for the measurement of free 125I radioactivity. The results are expressed as a percentage of the unbound 125I radioactivity to the total amount of 125I-albumin radioactivity instilled. These fluid samples always had <1% of unbound iodine present.

Determination of plasma concentration of epinephrine. In all experiments, the plasma concentration of epinephrine was measured. One milliliter of blood was collected in a heparinized tube just before the onset of hemorrhagic shock, at the end of the ischemic phase of hemorrhagic shock, and at the end of each experiment. Blood samples were immediately centrifuged at 14,000 rpm for 10 min at 4°C; 0.5 ml of plasma was transferred to an Eppendorf tube and quickly frozen to -70°C in acetone and dry ice. Samples were stored at -70°C until analyzed. Plasma and distal air space fluid samples were spiked with an internal standard and absorbed on activated alumina at pH 8.6. Epinephrine was eluted with 0.1 M perchloric acid, analyzed by reverse-phase HPLC with a C8 column, and measured by the amperometric method with an electrochemical detector. The recovery efficiency of plasma epinephrine was 60-70%. The correlation coefficient and detection limit of this method were 0.96 and 10 pg/ml, respectively.

Measurement of blood neutrophil count. On the day before the experiment, depletion of circulating neutrophils in vinblastine-pretreated rats was confirmed by evidence of <200 polymorphonuclear leukocytes/ml blood on a blood smear stained with modified May-Grünwald Giemsa.

Measurement of GSH and GSSG levels in lung epithelial lining fluid. For determining the levels of GSH in lung epithelial lining fluid, we used a variation of the HPLC method used by Martin and White (32). Briefly, bronchoalveolar lavage fluid was collected, and the cells were spun down at 14,000 rpm for 10 min at 4°C. Each sample was then extracted in 5% perchloric acid with 0.2 M boric acid and gamma -glutamyl-glutamate (10 µM) as an internal standard. Iodoacetic acid (final concentration 7 mM) was added, and the pH was adjusted to 9.0 ± 0.2. After incubation for 20 min to obtain S-carboxymethyl derivatives of thiols, dansyl chloride was added, and the samples were incubated for 24 h in the dark. The samples were then separated on an amine column with the solvents described by Reed et al. (49). Fluorescence detection was used for separation and quantification of the dansyl derivatives. The dilution of the lung epithelial lining fluid from the saline lavage was estimated by concomitant measurements of urea in the plasma and lavage fluid (50). Additionally, levels of GSSG were measured in the lung lavage fluid samples in parallel with the levels of GSH. From the lung lavage fluid, glutathione levels (GSH and GSSG) are expressed as a corrected concentration (in µM) in the epithelial lining fluid.

Myeloperoxidase activity assay. Myeloperoxidase (MPO) activity in the lung was measured with a modification of the method described by Mullane et al. (39). Briefly, lungs isolated from the rats were washed, blotted dry, and frozen in liquid nitrogen. After being weighed, the frozen lungs were homogenized in 2 ml of 50 mM phosphate buffer, pH 6.0, containing hexadecyltrimethylammonium bromide (0.5 g/100 ml). The samples were then freeze-thawed four times with liquid nitrogen and a 37°C water bath, sonicated, and centrifuged at 16,000 g for 20 min at 4°C. The supernatant was assayed in triplicate for activity in a hydrogen peroxide-o-dianisine buffer at 460 nm. The intra-assay coefficient of variation was 8 ± 2%. Results are expressed as units of MPO activity per gram of lung tissue.

Statistics

All the data are summarized as means ± SE. One-way analysis of variance and the Fisher's exact t-test were used to compare experimental with control groups. Nonparametric tests were used to compare plasma epinephrine values measured between experimental and control groups and concentrations of GSH and GSSG in alveolar epithelial lining fluid. A P value of <0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Prolonged Hemorrhagic Shock and Fluid Resuscitation

To induce severe hemorrhagic shock and maintain a systemic mean arterial pressure of 30-35 mmHg for 60 min, 35 ± 3% of the blood volume was withdrawn. This corresponded to the removal of 9.1 ± 1.4 ml of blood. The development of prolonged hemorrhagic shock resulted in severe systemic arterial hypotension (Fig. 2A) and metabolic acidosis (Fig. 2B). The final calculated base deficit of the arterial blood at the end of the ischemic phase of prolonged hemorrhagic shock was -16.5 ± 1.7 in hemorrhaged rats vs. -0.6 ± 0.6 in control rats (P < 0.05; Fig. 2C). The hemorrhaged rats were resuscitated over 30 min with approximately twice the volume of shed blood with a 4% bovine albumin solution (18.5 ± 0.9 ml). At the end of the studies, all physiological parameters in hemorrhaged rats were comparable to those measured in control rats.


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Fig. 2.   Effect of prolonged hemorrhagic shock on mean systemic arterial pressure (A), arterial pH (B), and arterial base deficit (C) in hemorrhaged and resuscitated () and control (open circle ) rats. Values are means ± SE. * P < 0.05 compared with control rats.

Prolonged hemorrhagic shock was associated with a significant increase in the endogenous release of catecholamines. There was a 20-fold increase in the epinephrine plasma levels at the end of the ischemic phase of hemorrhagic shock (2,414 ± 381 vs. 83 ± 15 pg/ml; P < 0.05; Table 1). Mean plasma levels of epinephrine remained significantly elevated until the end of the study in the hemorrhaged and resuscitated rats compared with those in the control rats (Table 1). There was also a significant threefold increase in lung MPO activity in rats that were hemorrhaged and fluid resuscitated compared with that in control rats (9.46 ± 1.02 vs. 3.22 ± 0.51 U/g lung tissue; P < 0.05).

                              
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Table 1.   Effect of prolonged hemorrhagic shock and fluid resuscitation on plasma epinephrine levels

However, in contrast to our results in rats that underwent short-term hemorrhagic shock and fluid resuscitation (37), there was no upregulation of alveolar liquid clearance after prolonged hemorrhagic shock. The final-to-initial distal air space unlabeled protein concentration was 1.27 ± 0.07 g/100 ml in control rats and 1.26 ± 0.07 g/100 ml in hemorrhaged rats (Table 2). Alveolar liquid clearance was comparable in control and hemorrhaged rats measured either with the nonlabeled bovine albumin (34 ± 4 vs. 36 ± 3%; Table 2) or with 125I-albumin (26 ± 0.07 vs. 27 ± 0.07%).

                              
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Table 2.   Final-to-initial alveolar fluid protein concentration and alveolar liquid clearance 5 h after prolonged hemorrhagic shock and fluid resuscitation

Hemorrhagic shock and fluid resuscitation were associated with a modest increase in the endothelial permeability to protein as indicated by the significant increase in extravascular plasma equivalents of the noninstilled lung in hemorrhaged rats compared with that in control rats (Table 3). Although the water-to-dry weight ratio of the noninstilled lung was not significantly different between hemorrhaged and control rats, the values measured after hemorrhagic shock and fluid resuscitation were significantly increased compared with those measured immediately after the ischemic phase of hemorrhagic shock (Table 4).

                              
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Table 3.   Extravascular plasma equivalents of noninstilled lung 5 h after prolonged hemorrhagic shock and fluid resuscitation


                              
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Table 4.   Extravascular water content of noninstilled lung 5 h after prolonged hemorrhagic shock and fluid resuscitation

Finally, there was no increase in the protein flux (125I-albumin) from the air spaces to the plasma. The percentage of instilled 125I-albumin recovered from the lungs and blood at the end of the experiments was comparable in hemorrhaged and control rats (Table 5).

                              
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Table 5.   Recovery of alveolar protein tracer 125I-albumin from lung and plasma 5 h after prolonged hemorrhagic shock and fluid resuscitation

Stimulation with Exogenous beta -Adrenergic Agonists After Prolonged Hemorrhagic Shock

Because there was no upregulation of alveolar epithelial liquid clearance 6 h after prolonged hemorrhagic shock and fluid resuscitation, the second objective was to maximize the response of beta -adrenergic receptors by the administration of exogenous beta -adrenergic agonists in hemorrhaged and fluid-resuscitated rats.

In rats that underwent prolonged hemorrhagic shock and fluid resuscitation, when epinephrine was administered intravenously (Fig. 3A) or in the distal air spaces (Fig. 3B), the expected increase in alveolar liquid clearance did not occur. Comparable results were obtained when salmeterol 10-5 M (n = 4 rats) was added to the protein solution instilled in the distal air spaces of the lung. In contrast, there was a significant increase in alveolar liquid clearance in control rats that were treated with epinephrine either intravenously (34 ± 4 vs. 44 ± 3%; Fig. 3A) or into the distal air spaces (34 ± 4 vs. 44 ± 3%; Fig. 3B). Comparable results were obtained when salmeterol (10-5 M; n = 3 rats) was instilled in the distal air spaces of control rats (34 ± 4 vs. 42 ± 2%).


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Fig. 3.   Alveolar liquid clearance in control and hemorrhaged rats. A: epinephrine administered intravenously (i/v). B: epinephrine (10-5 M) administered into distal air spaces. Values are means ± SE. Prolonged hemorrhagic shock (60 min) and fluid resuscitation did not upregulate alveolar liquid clearance in hemorrhaged rats that were given exogenous epinephrine. * P < 0.05 compared with control rats.

The severity of systemic arterial hypotension, metabolic acidosis, and calculated base deficit was comparable in rats that underwent prolonged hemorrhagic shock and were receiving exogenous beta -adrenergic agonists and in hemorrhaged rats that did not have any treatment.

Aministration of an Exogenous beta -Adrenergic Antagonist After Prolonged Hemorrhagic Shock

To determine whether prolonged hemorrhagic shock and fluid resuscitation affect basal and/or stimulated alveolar liquid clearance, a beta -adrenergic antagonist, propranolol (10-4 M), was added to the solution instilled into the distal air spaces of the lung of hemorrhaged rats. There was no further decrease in alveolar liquid clearance when epinephrine was given intravenously and propranolol (10-4 M) was added to the air space solution in in hemorrhaged and fluid-resuscitated rats (Fig. 3A). In control rats, the addition of propranolol to the protein solution instilled into the distal air spaces of the lung completely inhibited the upregulation of alveolar liquid clearance caused by the administration of intravenous epinephrine (Fig. 3A).

The severity of systemic arterial hypotension, metabolic acidosis, and calculated base deficit was comparable in rats that underwent prolonged hemorrhagic shock and were instilled with propranolol and in hemorrhaged rats that did not have any treatment.

Neutrophil Depletion Experiments

Because the administration of exogenous beta -adrenergic agonists did not stimulate alveolar fluid clearance, the third objective was to test the hypothesis that a neutrophil-dependent injury to the alveolar epithelium might account for the absence of upregulation of alveolar fluid clearance after prolonged hemorrhagic shock. Therefore, rats were neutrophil depleted with vinblastine given 4 days before hemorrhagic shock. There was a marked reduction in circulating neutrophils (<10 polymorphonuclear leukocytes/ml blood) in rats pretreated with vinblastine 4 days before hemorrhagic shock compared with that in control rats (3,800 ± 200 polymorphonuclear leukocytes/ml blood).

Neutrophil depletion with vinblastine 4 days before the onset of hemorrhagic shock completely restored the ability of the alveolar epithelium to upregulate alveolar liquid clearance in response to the instillation of epinephrine (10-5 M) into the distal air spaces of the lung (Fig. 4B). Comparable results were obtained from the first control group of rats that were neutrophil depleted but did not undergo prolonged hemorrhagic shock (Fig. 4A). To rule out an effect of vinblastine separate from its neutrophil depletion effect, rats that were given vinblastine 1 day before hemorrhagic shock and had a normal blood neutrophil count were studied. These rats did not show an upregulation of alveolar liquid clearance compared with rats that underwent a prolonged hemorrhagic shock (36 ± 1 vs. 36 ± 3%).


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Fig. 4.   Alveolar liquid clearance in control (A) and hemorrhaged (B) rats, both groups pretreated with vinblastine and instilled with epinephrine (10-5 M) into distal air spaces of lung. Values are means ± SE. Pretreatment with vinblastine restored ability of alveolar epithelium to upregulate fluid clearance in hemorrhaged rats that were stimulated with exogenous epinephrine. * P < 0.05 compared with control rats without air space epinephrine. ** P < 0.05 compared with hemorrhaged and resuscitated rats that were not pretreated with vinblastine.

Interestingly, neutrophil depletion with vinblastine 4 days before the onset of hemorrhagic shock was associated with a normalization of the endothelial permeability to protein. There was a significant decrease in extravascular plasma equivalents of the noninstilled lung in hemorrhaged rats that were depleted in neutrophils before the onset of shock compared with the values measured in hemorrhaged rats with a normal neutrophil count (Table 3).

There was no difference in the severity of hemorrhagic shock (mean arterial pressure, arterial pH, and base deficit; data not shown) in the plasma epinephrine levels (Table 1) and in the protein flux (125I-albumin) from the air spaces to the plasma (Table 5) between rats depleted of neutrophils 4 days before hemorrhagic shock and in rats with normal neutrophil counts. Comparable results were obtained for the rats that were given vinblastine 1 day before hemorrhagic shock (second control group).

Pretreatment With an Oxygen Radical Scavenger, NAC

Because neutrophil depletion with vinblastine before hemorrhagic shock was associated with a restoration of the response of the alveolar epithelium to beta -adrenergic agonists, the fourth objective of these studies was to test the hypothesis that oxidant-induced neutrophil-mediated injury to the alveolar epithelium could prevent upregulation of alveolar fluid clearance after prolonged hemorrhagic shock and fluid resuscitation. Therefore, GSH and GSSG levels were measured in alveolar epithelial lining fluid in control and hemorrhaged rats. In addition, GSH and GSSG were also measured in hemorrhaged rats that were pretreated with NAC, an oxygen radical scavenger.

Prolonged hemorrhagic shock significantly decreased GSH levels and the GSH-to-GSSG ratio in alveolar epithelial lining fluid (Table 6). However, there was a complete normalization of these glutathione levels after pretreatment with NAC.

                              
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Table 6.   Effect of prolonged hemorrhagic shock on GSH and GSSG levels in epithelial lining fluid 5 h after prolonged hemorrhagic shock and fluid resuscitation

Pretreatment with NAC 30 min before the onset of hemorrhagic shock completely restored the ability of the alveolar epithelium to upregulate alveolar fluid clearance in response to the instillation of epinephrine (10-5 M) in the distal air spaces (49 ± 3 vs. 36 ± 3%; Fig. 5B). Control studies of the effect of NAC in nonhemorrhaged rats treated with epinephrine showed no effect of NAC under these conditions (Fig. 5A).


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Fig. 5.   Alveolar liquid clearance in control (A) and hemorrhaged (B) rats that were pretreated with N-acetylcysteine (NAC) and instilled with epinephrine (10-5 M) into distal air spaces of lung. Values are means ± SE. Pretreatment with NAC restored ability of alveolar epithelium to upregulate fluid clearance in hemorrhaged rats that were instilled with exogenous epinephrine. * P < 0.05 compared with control rats without air space epinephrine. ** P < 0.05 compared with hemorrhaged and resuscitated rats that were not pretreated with NAC.

Interestingly, pretreatment with NAC 30 min before the onset of hemorrhagic shock was associated with a normalization of the endothelial permeability to protein. There was a significant decrease in extravascular plasma equivalents of the noninstilled lung in hemorrhaged rats that were pretreated with NAC 30 min before the onset of hemorrhagic shock compared with the values measured in hemorrhaged rats that were not pretreated with NAC (Table 3).

Systemic arterial hypotension, metabolic acidosis, calculated base deficit (data not shown), plasma epinephrine levels (Table 1), and protein flux (125I-albumin) from the air spaces to the plasma (Table 5) were comparable in rats that underwent prolonged hemorrhagic shock and were pretreated with NAC and in hemorrhaged rats that did not have any pretreatment.

XO Depletion Experiments

To exclude an effect of NAC that was not primarily related to its oxidant-scavenging properties, an additional series of experiments was done to inhibit the conversion from xanthine dehydrogenase to XO with a tungsten-enriched, molybdenum-deficient diet or an allopurinol-supplemented diet in hemorrhaged and fluid-resuscitated rats.

There was a restoration of the normal upregulation of alveolar liquid clearance in hemorrhaged rats that were fed either a tungsten-enriched or an allopurinol-supplemented diet before the onset of hemorrhagic shock (Fig. 6B). Control studies of the effect of XO depletion in nonhemorrhaged rats treated with epinephrine showed no effect of XO depletion under these conditions (Fig. 6A).


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Fig. 6.   Alveolar liquid clearance in control (A) and hemorrhaged (B) rats that were pretreated with a tungsten-enriched, molybdenum-deficient diet or an allopurinol-supplemented diet and instilled with epinephrine (10-5 M) into distal air spaces of lung. Values are means ± SE. Inhibition of conversion from xanthine dehydrogenase to xanthine oxidase by a tungsten-enriched molybdenum-deficient or an allopurinol-supplemented diet restores ability of alveolar epithelium to upregulate fluid clearance in hemorrhaged rats that were instilled with exogenous epinephrine. * P < 0.05 compared with control rats without air space epinephrine. ** P < 0.05 compared with hemorrhaged and resuscitated rats that were not fed tungsten-enriched molybdenum-deficient or allopurinol-supplemented diet.

Depletion of XO before the onset of hemorrhagic shock was associated with a normalization of the endothelial permeability to protein. There was a significant decrease in extravascular plasma equivalents of the noninstilled lung in hemorrhaged rats that were fed either a tungsten-enriched, molybdenum-deficient diet or an allopurinol-supplemented diet before the onset of hemorrhagic shock compared with the values measured in hemorrhaged nonpretreated rats (Table 3).

There was no difference in the severity of hemorrhagic shock (mean arterial pressure, arterial pH, and base deficit) and in the protein flux (125I-albumin) from the air spaces to the plasma (Table 5) between rats fed either a tungsten-enriched or an allopurinol-supplemented diet before the onset of hemorrhagic shock and in hemorrhaged rats that did not have any pretreatment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The overall objective of these studies was to determine the mechanisms that regulate fluid transport across the alveolar epithelium after prolonged hemorrhagic shock, one of the common causes of acute lung injury in humans (26). Because preservation of the capacity of the alveolar epithelium to actively remove fluid from the air spaces is critical for the survival of patients with acute lung injury (34), we developed an experimental animal model to investigate the effect of hemorrhagic shock on the function of the alveolar epithelium. The results of a previous study by Pittet et al. (44) indicate that the capacity of alveolar epithelium to remove fluid from the air spaces was maintained during the ischemic phase of hemorrhagic shock. In fact, there was an upregulation of the vectorial, sodium-dependent fluid transport across the alveolar epithelium secondary to the shock-mediated release of endogenous catecholamines (44). This protective mechanism against alveolar flooding was also sustained during the first 6 h after fluid resuscitation in the clinical setting of brief, severe hemorrhagic shock (37). However, the effect of prolonged hemorrhagic shock and fluid resuscitation on the function of the alveolar epithelium has not been studied.

Therefore, the first objective of these studies was to determine whether the endogenous release of catecholamines can upregulate alveolar fluid clearance after prolonged hemorrhagic shock and fluid resuscitation. The results indicate that although basal alveolar fluid clearance was maintained, there was no upregulation of alveolar fluid clearance across the alveolar epithelium in rats that underwent prolonged hemorrhagic shock (1 h) followed by fluid resuscitation despite a 20-fold increase in the epinephrine plasma levels at the end of the ischemic phase of hemorrhagic shock. Mean plasma levels of epinephrine remained significantly elevated until the end of the study in the hemorrhaged and resuscitated rats compared with those in control rats. In addition, there was no upregulation of alveolar liquid clearance when the response of beta -adrenergic receptors was maximized by the administration of exogenous beta -adrenergic agonists in hemorrhaged and fluid-resuscitated rats. Thus the results of these studies indicate a failure of the alveolar epithelium to respond to beta -adrenergic agonists, with the upregulation of vectorial fluid transport after prolonged hemorrhagic shock and fluid resuscitation.

The inability of the alveolar epithelium to respond to beta -adrenergic agonists could be explained by functional desensitization and downregulation of alveolar epithelial beta -adrenergic receptors after prolonged stimulation by those agonists. In vivo (59) and in vitro (28) exposure of lung epithelial cells to beta -adrenergic agonists can result in desensitization and downregulation of beta 2-adrenergic receptors by mechanisms involving an increase in the rate of receptor protein degradation. In contrast, the cAMP response to forskolin was unaffected by prior in vivo stimulation with beta -adrenergic agonists (59), and exposure to forskolin did not affect the beta 2-adrenergic-receptor density of cultured human airway epithelial cell monolayers (28). These results suggest that the activation of beta -adrenergic-receptor kinases is involved in this mechanism because these kinases require receptor occupancy to exert their effects, are highly expressed in the lung, and play an important role in mediating rapid catecholamine-induced beta -adrenergic-receptor desensitization in human airway epithelial cells (29). However, there are several reasons why it is unlikely that the exposure to beta -adrenergic agonists in these studies is responsible for the failure of the alveolar epithelium to upregulate alveolar fluid clearance in response to those agonists after prolonged hemorrhagic shock. First, catecholamine-induced downregulation of beta 2-adrenergic receptors was observed after a longer time exposure to beta -adrenergic agonists (24 h or more) than in our present experiments (6 h), although some beta 2-adrenergic-receptor desensitization may already occur in humans after a shorter exposure to beta -adrenergic agonists (42). Second, it has recently been reported (16) that the catecholamine-dependent upregulation of alveolar fluid transport is reversible when plasma epinephrine levels return to normal values after the administration of a constant intravenous epinephrine infusion in rats is stopped. Moreover, in that study (16), the administration of a second infusion of epinephrine 120 min after the first infusion was stopped and the plasma levels of epinephrine were normalized caused an upregulation of the vectorial fluid transport across the alveolar epithelium comparable with that observed after the first epinephrine infusion. Third, in the present experiments, control nonhemorrhaged rats that were stimulated with endogenous and/or exogenous epinephrine for 6 h had a significant upregulation of alveolar fluid clearance that was associated with a >20-fold increase in plasma levels of epinephrine (Table 1, Fig. 3). Fourth, Modelska et al. (37) previously reported that shock-mediated endogenous release of epinephrine caused a significant upregulation of alveolar fluid clearance in rats that underwent a brief period of severe hemorrhagic shock followed by fluid resuscitation. Therefore, these results and the data reported here indicate that desensitization of the beta -adrenergic receptors is not likely to be responsible for the failure of the alveolar epithelium to respond to beta -adrenergic agonists by upregulating alveolar fluid clearance. Interestingly, the results of a recent study (40) indicate that catecholamines and agents that increase cAMP cause a transient increase in chloride secretion by the alveolar epithelium of rabbits in vivo. The importance of this catecholamine-mediated chloride secretion by the alveolar epithelium in the present experimental model of hemorrhagic shock is unknown and needs to be further studied. Therefore, the mechanisms responsible for this lack of effect of beta -adrenergic agonists on alveolar fluid clearance after prolonged hemorrhagic shock are not completely understood and need to be elucidated in future studies.

We hypothesized that a neutrophil-dependent injury to the alveolar epithelium might account for the absence of upregulation of alveolar fluid clearance after prolonged hemorrhagic shock because there was a significant increase in the lung MPO activity of hemorrhaged and fluid-resuscitated rats compared with the values measured in control rats. Therefore, rats were pretreated with vinblastine, which caused a 99% depletion of circulating blood neutrophils. This pretreatment has previously been shown to markedly decrease the marginated pool of lung neutrophils as measured by MPO content in the lung and by histology (2). The results indicate that depletion of neutrophils by administration of vinblastine 4 days before the induction of prolonged hemorrhagic shock restored the ability of the alveolar epithelium to respond to beta -adrenergic agonists by upregulating alveolar fluid clearance (Fig. 4). This protective effect was not due to a nonspecific effect of vinblastine because it did not occur in hemorrhaged rats that were pretreated only 1 day before hemorrhagic shock and had a normal blood neutrophil count. Also, plasma epinephrine levels measured in hemorrhaged rats depleted of their neutrophils were in the same range as those measured in henorrhaged rats that had a normal neutrophil count, thus excluding that these results could simply be explained by a difference in plasma levels of epinephrine between both groups (Table 1). Thus these results indicate a neutrophil-dependent mechanism to explain the decrease in alveolar epithelial fluid transport after prolonged hemorrhagic shock.

In a model of hemorrhagic shock in rats that is comparable to the model used in the present study, neutrophil sequestration occurred in the lungs within 1 h after fluid resuscitation by an XO-related mechanism as indicated by a significant increase in lung MPO activity in hemorrhaged compared with control rats (3). Importantly, there was no increase in lung neutrophil sequestration in carefully matched sham control rats because anesthesia, catheter placement, and surgical stress could have caused a transient accumulation of neutrophils in the lungs (3). Moreover, neutrophil sequestration in the lung has been shown to contribute to diffuse lung endothelial injury induced by hemorrhagic shock (7, 17, 60). Also, the degree of activation of circulating neutrophils before the induction of shock is associated with the severity of shock-induced endothelial injury (6). However, the data in this study are the first results that demonstrate a link between neutrophil-mediated endothelial injury (as shown in this study by a modest increase in the endothelial permeability to protein) and a decrease in alveolar epithelial fluid transport that occurred without a major increase in alveolar epithelial permeability to protein. The data may be germane to human acute lung injury because both lung endothelial and epithelial injuries occur in patients with acute lung injury (5). Also, the severity of neutrophilic alveolar inflammation correlates with the mortality rate in a study of patients with acute lung injury (57). Finally, the functional status of the lung epithelium is likely to be a major factor that determines which patients develop alveolar flooding and prolonged respiratory failure after acute lung injury (33, 34).

The final objective of these studies was to test the hypothesis that neutrophil-induced injury to the fluid transport capacity of the alveolar epithelium was associated with an oxidative stress to the lung. Because glutathione has been shown to be one of the major oxidant scavengers in the alveolar epithelial lining fluid and a good marker for oxidative stress in the lung (51), GSH and GSSG levels were measured in the alveolar epithelial lining fluid of control and hemorrhaged rats. Prolonged hemorrhagic shock significantly decreased GSH levels and the GSH-to-GSSG ratio in alveolar epithelial lining fluid (Table 6), indicating the presence of an oxidative stress in the lung of hemorrhaged and fluid-resuscitated rats.

Evidence of oxidative stress has previously been reported in the lungs of animals and patients who died with clinical lung injury (21). Interestingly, the results of a morphological study (1) demonstrate that one of the predominant localizations of oxidizing species in the lung after ischemia-reperfusion was in alveolar epithelial type II cells. Reactive oxygen intermediates (e.g., H2O2) in vitro cause a significant depletion in intracellular GSH associated with an efflux of GSSG and a significant increase in the formation of protein-mixed disulfides (48). In humans, there is evidence of a significant reduction in the GSH-to-GSSG ratio in the epithelial lining fluid of patients with acute lung injury compared with normal subjects and patients with cardiogenic edema (15, 41). Similarly, plasma-oxidized glutathione is a marker of ischemia-reperfusion associated with single-lung transplantation (27). Moreover, there was a depletion in total GSH in the alveolar epithelial lining fluid of patients with a lung allograft (8). Thus the detection of a decrease in GSH and in the GSH-to-GSSG ratio in alveolar epithelial lining fluid of hemorrhaged rats indicates that the shock-mediated alveolar epithelial dysfunction was associated with the presence of oxidative injury to this barrier.

Therefore, to determine whether the inability of the beta -adrenergic-receptor agonists to upregulate vectorial fluid transport across the alveolar epithelium after prolonged hemorrhagic shock and fluid resuscitation was mediated through oxidant-mediated mechanisms, hemorrhaged rats were pretreated or treated with NAC, a glutathione precursor and an oxygen radical scavenger. The results indicate that pretreatment with NAC restored the ability of the alveolar epithelium to respond to beta -adrenergic agonists by upregulating alveolar fluid clearance in hemorrhaged and fluid-resuscitated rats. The restoration of the response of the alveolar epithelium to beta -adrenergic agonists after the administration of NAC was associated with a normalization of GSH levels and the GSH-to-GSSG ratio in alveolar epithelial lining fluid of hemorrhaged rats. Administration of NAC has previously been shown to decrease interleukin-1-induced neutrophil influx and lung leak in rats (30), to reduce endotoxin-induced neutrophil activation in sheep (9), to protect against phosgene lung injury in rabbits (55), and to reduce pulmonary endothelial cell damage induced by immune complexes deposition in rats (52). However, the present results are the first to demonstrate that NAC is able to correct the shock-mediated decrease in alveolar epithelial fluid transport function.

There are several mechanisms that may explain the protective effect of NAC against the shock-mediated oxidative injury to the alveolar epithelium. First, NAC may act by increasing intracellular and epithelial lining fluid GSH levels because it is a thiol-containing compound that is easily deacetylated to form cysteine, which can contribute to the synthesis of GSH (38). In fact, restoration of normal GSH levels in the alveolar epithelial lining fluid has been observed after intravenous administration of NAC in patients with idiopathic pulmonary fibrosis (35). Also, increases in intracellular and epithelial lining fluid GSH levels have been reported to protect in vitro rat alveolar type II cells against paraquat-induced cytoxicity (13, 23, 24) and in vivo preterm rabbits from oxidative lung injury (14). In addition to its direct extracellular antioxidant effect, GSH may protect signal transduction in alveolar epithelial type II cells by preventing oxidant-mediated inhibition of second messenger generation (inositol triphosphate and cAMP), events that are important for surfactant secretion and active ion transport by these cells (12). Also, NAC may have reversed the oxidant-mediated inhibition in ATP synthesis in alveolar epithelial cells (22). Second, NAC could act as a direct scavenger of oxygen radicals. NAC effectively inactivates hypochlorous acid and hydroxyl radical while minimally scavenging superoxide anion and H2O2 in vitro (4). Because all of these oxidants are produced by neutrophils, direct scavenging of neutrophil-derived oxygen radicals may contribute to protection of the alveolar epithelial transport function after the administration of NAC in hemorrhaged rats. Third, NAC may also inhibit oxidant-mediated nuclear factor (NF)-kappa B activation, cytokine and chemokine gene activation, and neutrophilic lung inflammation. Exposure of cells to reactive oxygen species has been shown to activate the transcription factor NF-kappa B (53). Moreover, NAC may block activation of NF-kappa B in vitro (53, 56) and in vivo after intraperitoneal administration of Escherichia coli endotoxin in rats (10). This mechanism may play an important role in our experimental animal model because it has recently been reported (31) that prolonged hemorrhagic shock (1 h) causes activation of NF-kappa B and a secondary increase in the expression of proinflammatory cytokines such as tumor necrosis factor-alpha , interleukin-1beta , and transforming growth factor-beta in lung mononuclear cells in mice.

To confirm the contribution of oxidative injury to the alveolar epithelium in these studies, we carried out additional experiments in which the conversion of xanthine dehydrogenase to reactive oxygen species-generating XO was inhibited by two different strategies, either by a tungsten-enriched diet or by pretreatment with allopurinol. Production of XO in hemorrhagic shock causes neutrophil sequestration in the lung (3) and activation of proinflammatory cytokine expression in lung mononuclear cells (54). Then, activated neutrophils induce conversion of xanthine dehydrogenase to XO by causing secretion of elastase in close proximity to lung endothelial cells (43). The results of these studies showed that the inhibition of XO formation by either of the inhibitors restored the ability of the alveolar epithelium to respond to beta -adrenergic stimulation after prolonged hemorrhagic shock (Fig. 6). Therefore, taken together, these data provide in vivo evidence for the presence of oxidative injury of the alveolar epithelium after prolonged hemorrhagic shock. Other investigators (20) have reported that oxidant injury to isolated alveolar epithelial type II cells decreases active transport of sodium and water across these epithelial monolayers. The present results may also have important clinical implications. It has recently been reported that plasma hypoxanthine levels, a prooxidant substrate for XO, are significantly higher in patients with acute lung injury than in mechanically ventilated control patients. The higher plasma hypoxanthine levels were associated with a loss of protein thiol groups and correlate with a poor outcome in these patients (47).

In summary, the present data provide the first in vivo evidence for a neutrophil-dependent decrease in alveolar epithelial fluid transport without a major alteration in the paracellular permeability to protein after prolonged hemorrhagic shock and fluid resuscitation. Because the expected catecholamine-mediated upregulation of alveolar liquid clearance was restored by normalization of the concentration of GSH in the alveolar epithelial lining fluid by NAC or by inhibition of the conversion of xanthine dehydrogenase to XO, these experiments also indicate that the decrease in the fluid transport capacity of the alveolar epithelium is mediated by an oxidant stress.


    ACKNOWLEDGEMENTS

We thank Ricardo Ciriales for technical assistance in carrying out these studies.


    FOOTNOTES

This work was primarily supported by National Heart, Lung, and Blood Institute Grant HL-51854. This project was also supported in part by a research grant (to J. F. Pittet) and a fellowship grant (to K. Modelska) from the American Lung Association, California.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. F. Pittet, Dept. of Anesthesia, Rm. 3C-38, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, CA 94110 (E-mail: jean_pittet{at}quickmail.ucsf.edu).

Received 20 August 1998; accepted in final form 27 January 1999.


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