Mechanisms of TNF-alpha stimulation of amiloride-sensitive sodium transport across alveolar epithelium

Norimasa Fukuda1, Christian Jayr1, Ahmed Lazrak2, Yibing Wang1, Rudolf Lucas3, Sadis Matalon2, and Michael A. Matthay1

1 Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130; 2 Department of Anesthesiology, Physiology, and Biophysics, University of Alabama, Birmingham, Alabama 35223; and 3 Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Because tumor necrosis factor (TNF)-alpha can upregulate alveolar fluid clearance (AFC) in pneumonia or septic peritonitis, the mechanisms responsible for the TNF-alpha -mediated increase in epithelial fluid transport were studied. In rats, 5 µg of TNF-alpha in the alveolar instillate increased AFC by 67%. This increase was inhibited by amiloride but not by propranolol. We also tested a triple-mutant TNF-alpha that is deficient in the lectinlike tip portion of the molecule responsible for its membrane conductance effect; the mutant also has decreased binding affinity to both TNF-alpha receptors. The triple-mutant TNF-alpha did not increase AFC. Perfusion of human A549 cells, patched in the whole cell mode, with TNF-alpha (120 ng/ml) resulted in a sustained increase in Na+ currents from 82 ± 9 to 549 ± 146 pA (P < 0.005; n = 6). The TNF-alpha -elicited Na+ current was inhibited by amiloride, and there was no change when A549 cells were perfused with the triple-mutant TNF-alpha or after preincubation with blocking antibodies to the two TNF-alpha receptors before perfusion with TNF-alpha . In conclusion, although TNF- alpha  can initiate acute inflammation and edema formation in the lung, TNF-alpha can also increase AFC by an amiloride-sensitive, cAMP-independent mechanism that enhances the resolution of alveolar edema in pathological conditions by either binding to its receptors or activating Na+ channels by means of its lectinlike domain.

tumor necrosis factor-alpha ; tumor necrosis factor receptor; patch clamp; alveolar epithelial cell; acute lung injury; pulmonary edema; pneumonia


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALVEOLAR EPITHELIAL FLUID transport is a fundamental mechanism for the resolution of alveolar edema (7, 12, 14, 35, 37, 38, 41, 42). Because of their location, alveolar epithelial cells are often exposed to increased intracellular and extracellular concentrations of reactive oxygen and nitrogen species and inflammatory cytokines such as tumor necrosis factor (TNF)-alpha . Several studies (8, 45, 46, 48, 52, 55) have demonstrated that TNF-alpha may have either a deleterious or a protective effect during the inflammatory response after infection (53, 54). Rezaiguia et al. (44) reported that acute Pseudomonas aeruginosa pneumonia resulted in a marked upregulation of the rate of net alveolar epithelial Na+ and fluid clearance in rats, an effect that depended on TNF-alpha stimulation of alveolar fluid transport. A recent study (10) also reported that TNF-alpha mediates upregulated alveolar fluid clearance (AFC) in rats with septic peritonitis. TNF-alpha may also upregulate nitric oxide production by inflammatory cells (1, 22, 28, 50), an effect that could have several effects on ion transport (15, 16, 23).

To investigate the mechanisms by which TNF-alpha upregulates AFC, we carried out both in vivo and isolated cell studies. First, using intact rats, we determined whether intratracheal instillation of TNF-alpha increased AFC by amiloride-sensitive mechanisms in rats and whether the TNF-alpha effect in rats was inhibited by a beta -antagonist or accelerated by a beta -agonist. Also, to gain insight into how TNF-alpha may upregulate AFC, we carried out experiments in rats with a triple-mutant TNF-alpha that lacks the lectinlike region of the molecule that is responsible for the membrane conductance-activating effect of the molecule (26, 33); the triple mutant also has a modest decrease in binding to the two known TNF-alpha receptors TNFRI and TNFRII (32). To elucidate the mechanisms of TNF-alpha action, we measured Na+ currents across A549 cells, a human alveolar epithelial cell line that possesses many characteristics of type II cells, including Na+-selective amiloride-sensitive channels (31), patched in the whole mode. These measurements were performed before and after perfusion of A549 cells with TNF-alpha , TNF-alpha plus amiloride, or the triple-mutant TNF-alpha and across A549 cells preincubated with blocking antibodies to TNFRI and TNFRII before perfusion with TNF-alpha .


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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Male Sprague-Dawley rats (n = 46; 250-350 g) were used for all animal experiments. The rats were housed in air-filtered, temperature-controlled units with food and water. All procedures were approved by the University of California, San Francisco Committee on Animal Research.

Surgical Preparation for AFC Measurements

Rats were anesthetized with pentobarbital sodium (50-100 mg/kg ip). A tracheostomy was done, and a 0.2-mm-internal diameter endotracheal tube (PE-240, Clay Adams, Becton Dickinson, Parsippany, NJ) was inserted. The rats were maintained in the right decubitus position and ventilated (Harvard Apparatus, Millis, MA) with 100% O2, peak airway pressure of 12-15 cmH2O, and positive end-expiratory pressure of 3 cmH2O. The respiratory rate was adjusted to maintain arterial PCO2 between 35 and 45 mmHg. A catheter was inserted into the right carotid artery to monitor systemic blood pressure and obtain blood samples. Body temperature was always kept constant at 38°C by placing the animals on a thermostatically controlled pad. This protocol was according to previous studies by Jayr et al. (27) and Rezaiguia et al. (44).

Preparation of the Instillate

A 5% isosmolar bovine serum albumin (BSA; Sigma, St. Louis, MO) solution with Ringer lactate was prepared according to previous reports (24, 30, 49). We added 1 mg of anhydrous Evans blue dye and 0.5 µCi of 125I-labeled human serum albumin (Merck-Frosst, Montreal, PQ) to the instillate. In some studies, 10-3 M amiloride (Sigma), 10-4 M terbutaline (Sigma), 10-4 M propranolol (Sigma), wild-type TNF-alpha (5 µg/rat; Innogenetics, Ghent, Belgium), or a triple-amino acid lectin-deficient mutant of TNF-alpha (5 µg/rat) was added into the instillate. A sample of the instillate was obtained for measurement of total protein, 125I radioactivity, and water-to-dry weight ratio.

TNF-alpha

Wild-type mouse TNF-alpha and the triple-amino acid mutant mouse TNF-alpha were provided by Innogenetics. The triple-mutant TNF-alpha was generated by amino acid replacement of residues Thr104, Glu106, and Glu109 by alanine in the lectinlike domain of the molecule situated in the tip region the TNF trimer. This portion of the molecule is responsible for the Na+ channel-activating capacity (26), and this mutant was originally developed to study the role of the lectinlike domain of TNF-alpha (32). Also, the mutant does have a 5-fold reduced receptor affinity for TNFR1 and a 10-fold reduced receptor affinity for TNFR2 (32).

General Protocol

A 1-h baseline period of stable blood pressure and heart rate was required before alveolar fluid instillation. 131I-labeled albumin (3 µCi) was injected intravenously as a vascular tracer 15 min before instillation. The vascular tracer was used to calculate the flux of plasma protein into the airspaces (27). Blood samples were obtained every 30 min during the experiment for 131I-albumin and 125I-albumin radioactivity and arterial blood gas determinations. We instilled 6 ml/kg of isosmolar fluid into both lungs. At the end of studies (60 min), the rats were exsanguinated and the lungs were removed through a midline sternotomy. An alveolar fluid sample (0.1-0.2 ml) was aspirated with a 3-ml syringe and Silastic tubing that was passed into a wedged position in both lungs. Total protein and radioactivity of the alveolar fluid sample were measured. The lungs were homogenized for extravascular lung water measurements and radioactivity counts.

Specific Protocols: Rat Studies

Basal alveolar fluid clearance was determined in group 1 (n = 4 rats). The effects of TNF-alpha (5 µg/rat; n = 7 rats) and (1 µg/rat; n = 4 rats) and the triple-mutant TNF-alpha (5 µg/rat; n = 5 rats) on AFC were studied in group 2. The effect of amiloride (10-3 M; n = 5 rats) on basal AFC and the combined effect of amiloride (10-3 M) and wild-type TNF-alpha (5 µg/rat; n = 6 rats) on AFC were studied in group 3. The effect of terbutaline (10-4 M; n = 5 rats) on basal AFC and the combined effect of terbutaline (10-4 M) and wild-type TNF-alpha (n = 5 rats) on AFC were studied in group 4. The effect of propranolol (10-3 M; n = 5 rats) on TNF-alpha -stimulated was studied in group 5.

Measurement of AFC

According to prior studies by our laboratory (7, 18, 27, 37, 41, 44), AFC was estimated by measuring the increase in the final concentration of the alveolar protein tracer compared with the initial instilled tracer protein concentrations. We subtracted the dry weight of the added protein in the lung water calculation from the final alveolar sample.

Patch Clamp

Cell line and culture methods. A549 cells were purchased from American Type Culture Collection (Manassas, VA) in the 76th passage. They were suspended in DMEM-F-12 medium (Cellgro) supplemented with 1% penicillin-streptomycin and 10% fetal calf serum, plated on plastic tissue culture flasks (Corning Glass Works, Corning, NY), and placed in an incubator in 21% O2, 5% CO2, and balance N2 at 37°C and 100% humidity. All experiments were carried out on cells between the 78th and the 97th passages.

Electrophysiology, patch-clamp recording, and analysis. Macroscopic currents were recorded from A549 cells in the whole cell recording mode of the patch-clamp technique (23).

Between 24 and 36 h before any electrophysiological measurements, the A549 cells were lifted from the tissue plates by treatment with 2.5% trypsin-EDTA (Sigma) for 3-6 min at 37°C and then seeded on 12-mm-diameter glass coverslips in DMEM-F-12 medium. The coverslip was rinsed with standard external solution (SES; 310-320 mosM) just before the onset of the measurements. The composition of SES was 145 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 2.0 mM MgCl2, 5.5 mM glucose, and 10 mM HEPES, pH 7.4 (with NaOH). The cells were then transferred to the recording chamber that was mounted on the stage of an inverted microscope (IMT-2, Olympus, Tokyo, Japan) for patch-clamp recordings.

The pipettes were made from LG16-type capillary glass (Dagan, Minneapolis, MN) with a vertical puller (model PB-7, Narishige, Japan). The intrapipette solution consisted of 135 mM potassium methylsulfonic acid, 10 mM KCl, 6 mM NaCl, 1.0 mM Mg2ATP, 2.0 mM Na3ATP, 10.0 mM HEPES, and 0.5 mM EGTA, pH 7.2 (with 1 N KOH), at 22°C (standard internal solution; 300 mosM). The pipette resistance varied from 3 to 5 MOmega when back-filled with standard internal solution. The pipette offset potential was corrected just before gigaseal formation. Series resistance and capacitance transients were compensated for with the patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA). The junction potentials between the pipette solution and the bath solution at the tip of the pipette and between the perfusing solution and the agar bridge were corrected for as described in a prior report (5). The preparation was grounded with a Ag-AgCl electrode connected to the bath via an agar bridge (2%). The solution in the recording chamber (400 µl) was changed with a gravity-driven perfusion system.

The cell membrane potential was held at -40 mV during all whole cell recordings. Inward currents were elicited by altering the membrane potential from the holding value (-40 mV) by -100 mV for 450 ms every 5 s with the Clampex program (pCLAMP, Axon Instruments). The currents were digitized at 5 kHz with a digital-to-analog, analog-to-digital converter (DigiData 1200A, Axon Instruments), filtered through an internal four-pole Bessel filter at 2 kHz, and stored on the hard disk of a computer.

After baseline currents were recorded, A549 cells, while still patched in the whole cell mode, were perfused with SES containing either 10 or 120 ng of wild-type or triple-mutant TNF-alpha for 2-3 min, which resulted in a steady-state increase in inward Na+ currents. The cells were then perfused with either SES alone or SES containing 10 µM amiloride for 5-8 min. Inward Na+ currents were measured continuously throughout this period. To further investigate the potential mechanisms by which TNF-alpha increased Na+ currents, A549 cells, patched in the whole cell mode, were incubated with SES containing anti-human soluble (s) TNFRI and sTNFRII antibodies (5 ng each; R&D Systems) or an equivalent amount of nonspecific mouse IgG for 3 min. The specificity of the antibodies has been demonstrated (38). They were then perfused with the SES containing sTNFRI, sTNFRII, and 120 ng/ml of TNF-alpha . Inward Na+ currents were measured as above.

Statistics

All data are means ± SD unless otherwise noted. AFC data were analyzed by one-way ANOVA followed by Mann-Whitney U-test post hoc. Time-dependent effects were analyzed by repeated-measures ANOVA followed by Student-Newman-Keuls test post hoc. Significance was defined as P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Wild-Type TNF-alpha and Amiloride on AFC in Rats

Wild-type TNF-alpha (5 µg/rat) increased AFC by 67% (P < 0.05) over 1 h in the ventilated rats. Amiloride decreased basal AFC by 41% (P < 0.05) and prevented the TNF-alpha -induced upregulation of AFC (P < 0.05; Fig. 1). A lower dose of TNF-alpha (1 µg/rat) did not increase AFC (data not shown).


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Fig. 1.   Effect of wild-type tumor necrosis factor (TNF)-alpha (5 µg/rat) and amiloride on alveolar fluid clearance in rats. Values are means ± SD; n, no. of rats. * P < 0.05 compared with control. ** P < 0.05 compared with TNF-alpha group.

Effect of beta -Adrenergic Stimulation and Blockade on AFC During TNF-alpha Instillation in Rats

Terbutaline alone increased AFC by 65% in rats (P < 0.05), similar to the effect of wild-type TNF-alpha . However, the combination of terbutaline and TNF-alpha had no additional effect on AFC (Fig. 2). Propranolol, a beta -adrenergic receptor antagonist, did not decrease the TNF-alpha -stimulated AFC in rats (Fig. 2)


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Fig. 2.   Effect of wild-type TNF-alpha on alveolar fluid clearance in rats. TNF-alpha , amiloride, terbutaline, and propranolol were combined with the instilled solution. Values are means ± SD; n, no. of rats. * P < 0.05 compared with control.

Effect of Wild-Type TNF-alpha and Triple-Mutant TNF-alpha on AFC in Rats

Wild-type TNF-alpha significantly increased AFC by 67% in ventilated rats (P < 0.05), but the triple-mutant TNF-alpha did not upregulate AFC (Fig. 3).


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Fig. 3.   Effect of triple-mutant TNF-alpha compared with wild-type TNF-alpha on alveolar fluid clearance. Data are means ± SD; n, no. of rats. * P < 0.05 compared with control and triple-mutant TNF-alpha .

Patch-Clamp Experiments on A549 Cells With Wild-Type and Triple-Mutant TNF-alpha

Whole cell inward current was elicited by the application of -100 mV to cells patched in the whole cell mode and held at -40 mV. Perfusion of A549 cells with 10 ng/ml of TNF-alpha did not increase the baseline current (data not shown). However, perfusion with 120 ng/ml of TNF-alpha (n = 6 cells) resulted in a large increase in the inward Na+ current, starting within 30 s from the start of perfusion (Fig. 4A). This increase was sustained when the cell was reperfused with SES alone (Fig. 4A) but was completely abolished when amiloride (10 µM) was added into the perfusion medium (Fig. 4B). On the other hand, perfusion with the triple-mutant TNF-alpha (120 ng/ml) did not increase the inward currents (Fig. 5). Perfusion with TNF-alpha in the presence of two blocking antibodies to TNFRI and TNFRII failed to increase the inward current (Fig. 6). In contrast, when equivalent amounts of nonimmune IgG were substituted for the primary antibodies, TNF-alpha rapidly increased the whole cell current (Fig. 6). Mean values for the TNF-alpha -elicited increases in inward currents for the various experimental conditions shown above are provided in Fig. 7, with a P value of <0.005 for the differences.


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Fig. 4.   A: effect of wild-type TNF-alpha on Na+ current in a A549 cell patched in the whole cell mode. The pipette was filled with standard internal solution (see MATERIALS AND METHODS for details). The cell was held at -40 mV, and an inward current (negative current) was elicited by an application of a -100-mV pulse. The cell was perfused with either standard external solution (SES) or SES containing TNF-alpha (120 ng/ml) as indicated. B: typical experiment that was repeated 6 times with the same experimental conditions as in A except that the A549 cell was perfused with SES containing 10 µM amiloride after perfusion with TNF-alpha .



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Fig. 5.   Effect of triple-mutant TNF-alpha on Na+ current in an A549 cell patched in the whole cell mode. The experimental conditions are described in Fig. 4A except that this A549 cell was perfused with SES containing the triple-mutant TNF-alpha (120 ng/ml). In sharp contrast to the result in Fig. 4A, the inward Na+ current was not altered during perfusion. Results represent a typical experiment, which was repeated 6 times.



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Fig. 6.   A: perfusion of A549 cells with wild-type TNF-alpha in the presence of blocking antibodies to its receptors did not increase Na+ currents. An A549 cell was patched in the whole cell mode as described in Fig. 4A and was perfused with SES containing anti-human soluble TNF receptor types I and II (sTNFRI and sTNFRII, respectively) antibodies (5 ng each) for 10 min. When a stable value of the inward Na+ current was obtained, the solution was switched to one containing sTNFRI, sTNFRII, and 120 ng/ml of TNF-alpha for ~5 min. No change in the inward current was observed. In contrast, when equivalent amounts of nonimmune IgG were substituted for the primary antibodies (B), TNF-alpha rapidly increased the whole cell current. At the end of this period, the cell was perfused with SES containing 10 µM amiloride, which blocked the existing Na+ current. Results represent a typical experiment, which was repeated 6 times.



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Fig. 7.   Mean values of inward Na+ currents elicited after perfusion with wild-type TNF-alpha (120 ng/ml), TNF-alpha (120 ng/ml) in the presence of blocking antibodies to TNFRI and TNFRII (5 ng/ml each), or triple-mutant TNF-alpha (120 ng/ml). Values are means ± SE; n = 6 cells. * P < 0.005 compared with other groups.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The prior study by Rezaiguia et al. (44) indicated that AFC was upregulated by a TNF-alpha -dependent mechanism in acute bacterial pneumonia in rats. A more recent article (10) confirmed that TNF-alpha could augment alveolar epithelial fluid transport in a model of septic peritonitis in rats. However, the mechanism by which TNF-alpha upregulates AFC has not been explored. Our objective was to study the mechanisms that could account for the TNF-alpha -induced upregulation of alveolar fluid transport in both intact rats and isolated human alveolar A549 epithelial cells. Some investigators (3, 4, 29, 33) have reported that TNF-alpha can create ion channels by insertion into the plasma membrane as reported in human U937 histiocytic lymphoma cells. In those studies, the effect was enhanced in the presence of a low pH, perhaps because TNF-alpha binding to the TNFRs was markedly decreased in a low pH environment (3, 4). However, van der Goot et al. (51) reported that membrane insertion is not sufficient for the membrane conductance activating effect of TNF-alpha . In the current study, we used a triple-amino acid mutant that does not contain the lectinlike domain of TNF-alpha that is responsible for its membrane conductance effects; the mutant also has a modest decrease in receptor binding (32).

The major findings of these studies can be summarized as follows. 1) Wild-type TNF-alpha upregulated AFC in rats, a finding consistent with the prior study by Rezaiguia et al. (44) in rats with pneumonia as well as with the recent study of sepsis (10). 2) Propranolol, a beta -adrenergic antagonist, did not decrease TNF-alpha -stimulated AFC in rats. 3) No additional upregulation occurred with the combination of TNF-alpha and a beta -adrenergic agonist. 4) Amiloride blocked the TNF-alpha -stimulated AFC in rats. 5) The triple-mutant TNF-alpha did not stimulate AFC in rats. 6) In patch-clamp studies of the human alveolar epithelial A549 cell line, TNF-alpha stimulated Na+ influx in A549 cells, an effect that was inhibited by amiloride. The triple-mutant TNF-alpha did not induce Na+ current in the A549 cells, and the TNFR blocking antibodies abolished the effect of TNF-alpha .

Because the effect of TNF-alpha occurred within 30 s from the onset of perfusion in the A549 cells and within 1 h from its instillation in the distal airspaces of the rat, it is apparent that the primary mechanism does not depend on a transcriptional effect of TNF-alpha . Because the TNF-alpha effect was inhibitable by amiloride in both the intact rat lung studies and the isolated human A549 epithelial cells, the primary pathway for augmented fluid transport in lung epithelium depends on Na+ transport. We then tried to determine whether the TNF-alpha effect could be mediated by an effect on membrane conductance by a receptor-independent mechanism. In a prior study (26), peritoneal macrophages from TNFR double-knockout mice still showed an increase in ion channel activity in the presence of wild-type TNF-alpha , and this activity was amiloride sensitive. Also, a peptide mimicking the lectinlike domain, with no binding to the two TNFRs, can still trigger increases in membrane conductance and also be inhibited by amiloride (33). Because the triple-mutant TNF-alpha did not stimulate AFC in the rat studies, it seemed plausible that the effect of TNF-alpha was mediated by receptor-independent mechanisms in the lung.

But this interpretation could be incomplete, because the TNF-alpha mutant retains some receptor affinity, although the affinity is decreased 5-fold for TNFR1 and 10-fold for TNFRII (32). Thus the results of the in vivo studies could be consistent with a receptor-dependent effect, a receptor-independent effect, or both.

Interestingly, the patch-clamp studies in the A549 cells also demonstrated a rapid amiloride-inhibitable uptake of Na+ in the presence of wild-type TNF-alpha , consistent with the data in the intact rat lung studies. Also, the triple-mutant TNF-alpha did not increase Na+ influx in the A549 cells, also consistent with the results of the rat studies. Therefore, we took advantage of the availability of specific human TNFRI and TNFRII blocking antibodies to determine whether the effect of TNF-alpha in the A549 cells was receptor mediated. The results provided direct evidence in these cells for a receptor-dependent effect. Several studies (39, 46-48, 50) have shown that TNFRI is expressed in the lung in airway epithelium and alveolar epithelium and that both receptors exist in A549 cells.

However, the mechanism by which TNF-alpha increases Na+-dependent AFC in vivo may be considerably more complicated and may involve multiple pathways. TNF-alpha could stimulate the release of other mediators in vivo, such as transforming growth factor-alpha (9, 52), which has been shown to rapidly upregulate AFC in rats (17). Furthermore, it is certainly plausible that receptor-independent effects may occur in vivo in the lung and that some of the TNF-alpha effects on enhancing alveolar fluid reabsorption across the alveolar epithelium could be secondary to direct effects on the cell membrane.

The combination of TNF-alpha and a beta -adrenergic agonist, terbutaline, did not have an additive effect on increasing AFC in the TNF-alpha -instilled rats. Also, propranolol, a beta -adrenergic antagonist, did not inhibit the TNF-alpha -induced upregulation of alveolar epithelial fluid transport in rats. These results are similar to the findings after instillation of either endotoxin (21) or bacteria (44) into rat lungs. Taken together, the data indicate that TNF-alpha -induced alveolar epithelial fluid transport is not mediated by an endogenous release of epinephrine, a finding that is in agreement with the results of a recent peritonitis study in rats (10).

Which signaling mechanism mediated the TNF-alpha -induced Na+ uptake? TNF-alpha is known to decrease intracellular cAMP (13, 40), and the recent study by Börjesson et al. (10) showed that TNF-alpha upregulation of AFC in rats with peritonitis was associated with no change in cAMP in the lung. Therefore, enhanced Na+ and fluid transport from TNF-alpha probably does not depend on a cAMP-mediated process. Interestingly, G proteins can mediate several effects of TNF-alpha (25), and a G protein has been shown to contribute to Na+ transport in fetal alveolar type II cells (19, 20, 30, 34). Also, pertussis toxin can inhibit transepithelial Na+ transport (2). However, there is no clear evidence for the association of G protein with an immunopurified alveolar type II cell Na+ transport at this time (6). Thus further investigation will be needed to elucidate the mechanisms of TNF-alpha signaling in alveolar epithelial cells.

TNF-alpha has been shown in several studies (11, 49) to increase lung and systemic vascular permeability. Also, in vitro studies indicated that TNF-alpha can alter short-circuit current across cultured alveolar type II cells (56), and TNF-alpha decreases surfactant protein B expression (43). Thus TNF-alpha can have a deleterious effect on the lung endothelium and the alveolar epithelium, although the data in this study and other studies (10, 44) documented a beneficial effect of TNF-alpha on lung fluid balance by upregulating alveolar epithelial fluid transport.

In summary, TNF-alpha upregulates alveolar epithelial Na+ and fluid transport by an amiloride-sensitive, catecholamine-independent mechanism as demonstrated by studies in both intact rats and isolated human A549 alveolar epithelial cells. In the isolated cell studies, the effect was mediated by a TNFR-dependent process, although the intact rat studies do not clearly distinguish between receptor-dependent and -independent effects of TNF-alpha . Overall, these results provide further evidence for a beneficial effect of TNF-alpha on lung fluid balance that may be germane to clinically important pathological conditions such as pneumonia (44) or peritonitis (10).


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-51854 and HL-31197.


    FOOTNOTES

Address for reprint requests and other correspondence: M. A. Matthay, Cardiovascular Research Institute, Univ. of California, 505 Parnassus Ave., HSW-825, San Francisco, CA 94143-0130 (E-mail: mmatt{at}itsa.ucsf.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 13 April 2000; accepted in final form 18 December 2000.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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