Children's Hospital Oakland Research Institute, Oakland, California 94609
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ABSTRACT |
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Acid secretion and proton
conductive pathways across primary human airway surface
epithelial cultures were investigated with the pH stat method in Ussing
chambers and by single cell patch clamping. Cultures showed a basal
proton secretion of 0.17 ± 0.04 µmol · h1 · cm
2, and
mucosal pH equilibrated at 6.85 ± 0.26. Addition of histamine or
ATP to the mucosal medium increased proton secretion by 0.27 ± 0.09 and 0.24 ± 0.09 µmol · h
1 · cm
2,
respectively. Addition of mast cells to the mucosal medium of airway
cultures similarly activated proton secretion. Stimulated proton
secretion was similar in cultures bathed mucosally with either NaCl
Ringer or ion-free mannitol solutions. Proton secretion was potently
blocked by mucosal ZnCl2 and was unaffected by mucosal bafilomycin A1, Sch-28080, or ouabain. Mucosal amiloride
blocked proton secretion in tissues that showed large
amiloride-sensitive potentials. Proton secretion was sensitive to the
application of transepithelial current and showed outward
rectification. In whole cell patch-clamp recordings a strongly
outward-rectifying, zinc-sensitive, depolarization-activated proton
conductance was identified with an average chord conductance of
9.2 ± 3.8 pS/pF (at 0 mV and a pH 5.3-to-pH 7.3 gradient). We
suggest that inflammatory processes activate proton secretion by the
airway epithelium and acidify the airway surface liquid.
primary airway cultures; JME airway cells; pH stat; patch clamp; Ussing chamber
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INTRODUCTION |
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THE MUCOSA OF THE
AIRWAY SURFACE epithelium is lined with a thin layer of fluid
called the airway surface liquid (ASL). The composition of the ASL
affects its physiological functions, the most important of which are
removal of inhaled particles and antimicrobial activity
(36). Active transport of Na+ and
Cl by the airway epithelium have been well characterized
and recognized as critical determinants of ASL composition and depth.
In contrast, the regulation of the H+ concentration in the
ASL has received little attention. Acidic luminal pH has been shown to
inhibit ciliary beating (4) and to cause
bronchoconstriction (1), cough (39),
loosening of the epithelial cells from one another (13),
and detachment from the basement membrane (17). Acidic ASL
has been implicated in airway diseases such as asthma (19,
29) and cystic fibrosis (CF) (33). Although the
airway epithelium has been shown to secrete HCO
The concentration of HCO channel (31). Jayaraman et al.
(22) estimated a HCO
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METHODS |
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Cell cultures. Human tracheal primary cultures were isolated and cultured as previously described (40). In brief, strips of epithelium were removed from the underlying tissues and treated with protease overnight. The resulting isolated, dispersed cells were plated on permeable filter supports (Snapwell, 0.4-µm pore size, 1-cm2 area; Corning Costar, Cambridge, MA) precoated with human placental collagen (15 µg/cm2) at a density of ~106 cells/cm2. Cells were grown in DMEM-F12 culture medium supplemented with 2% Ultroser G (Biotechnics, Paris, France) and antibiotics. Tracheal sheets were grown to confluence in an air-liquid interface in a tissue culture incubator gassed with 5% CO2 and air. The cystic fibrosis JME/CF15 airway cell line (23) was cultured in a DMEM-F-12 mixture supplemented with 10% fetal bovine serum and (per ml) 5 µg of insulin, 0.5 µg of hydrocortisone, 10 ng of epidermal growth factor, 5 ng of transferrin, 1.3 ng of triiodothyroxine, 43 ng of adenine, and 1 µg of epinephrine. For patch clamping, cells were seeded at low density on cover glasses. The human mast cell line HMC-1 was kindly provided by Dr. J. H. Butterfield and was cultured as described previously (2).
Quantification of proton secretion.
Proton secretion was measured with the pH stat titration method in an
Ussing chamber (14). Cultures (1-cm2 exposed
area) were mounted in an Ussing chamber (Physiologic Instruments, San
Diego, CA) and, unless otherwise described, bathed serosally with
HEPES-buffered solution and mucosally with buffer-free solution (3 ml
each). Solutions were constantly gassed with oxygen and were nominally
free of HCO
Whole cell patch-clamp recordings.
Cells were whole cell patch-clamped after 2 days in culture as
previously described (20) on a temperature-controlled
stage (35-37°C) of an inverted microscope. Cells were bathed in
(in mM) 100 HEPES, 85 gluconic acid, 100 NMDG, 2 Ca gluconate, 1 Mg gluconate, and 10 glucose, pH 7.3. The pipette was filled with (in mM)
100 2-(N-morpholino)ethanesulfonic acid (MES), 81.6 gluconic acid, 70 NMDG, 10 NMDG-EGTA, 1 glucose, 1 MgCl2, 3.3 Mg-ATP, and 0.07 Li-GTP, pH 5.3. The bath electrode was made with the
pipette-filling solution (but ATP/GTP free) and a 3% agar bridge.
Junction potentials were measured, zeroed, and carefully observed for
stability. With these solutions, pipette resistance was ~5 M. Only
seals >50 G
were used for recordings. The access resistance
(Ra) and the cell membrane capacitance
(Cm) were determined by fitting the current
transients caused by a 10-mV voltage pulse with a single exponential.
Measured Ra was 15.4 ± 1.8 M
(n = 13), and Cm was 29.1 ± 4.3 pF. Current-voltage step protocols from
80 mV to +60 mV were
applied, and resulting step currents were recorded. Whole cell
conductance was calculated as the chord conductance at 0 mV. When the
specific membrane conductance (Gm in pS/pF) was
calculated, whole cell conductance was corrected for
Ra and normalized to Cm.
Drugs. Stock solutions of histamine (free base, 10 mM), ATP (Na salt, 50 mM), and ZnCl2 (1 mM) were prepared in NaCl Ringer solution, and pH was adjusted to 7.3. Amiloride stock was prepared at 10 mM in water, and pH was adjusted to 7.3. 3-(Cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo- [1,2a]-pyridine (Sch-28080) was kindly provided by Dr. T. E. Machen (Univ. of California, Berkeley) and prepared as a 5 mM stock in ethanol. Bafilomycin A1 (500 µM) and ouabain (100 mM) were prepared as stocks in dimethyl sulfoxide.
Statistics. Data are given as original values or as means ± SE; n is the number of cultures tested. Unpaired t-tests were used to test for statistical difference (P < 0.05) between means, and one-sample t-tests were used to test whether responses were significantly different from zero.
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RESULTS |
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Proton secretion across human airway cultures was investigated in
HCO1 · cm
2
(n = 26). Basal Vt was
15.6 ± 4.1 mV, and Rt was 1,016 ± 137
· cm2. Figure
1 shows an example of a measurement of
mucosal pH and determination of the basal rate of acidification.
Initially, the pH was titrated two times with NaOH to ~7.3 to
calculate the rate of acidification. When no NaOH was added, the pH
reached an equilibrium at 6.85 ± 0.26 (n = 5)
after 41 ± 9 min.
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Addition of histamine or ATP (100 µM each) to the mucosal bath
potently stimulated H+ secretion (Fig.
2, A and B). Both
agents typically caused a fast initial rise followed by a slow decay of
JH. Figure 2C shows the average peak effects of
mucosal addition of histamine and ATP. In contrast, when
histamine or ATP was added to the serosal bath, JH was
unchanged (not shown). We also tested 50 µM serotonin and 20 µM
forskolin, which showed no significant effects on JH
[serotonin, +0.04 ± 0.05 µmol · h1 · cm
2
(n = 3); forskolin, +0.01 ± 0.10 µmol · h
1 · cm
2
(n = 3)].
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To identify the mechanism(s) in the apical membrane responsible for the
H+ secretion we tested known blockers of H+
transporters. Cultures were stimulated with a combination of histamine
and ATP, which we found to result in more sustained and stable
responses compared with application of the single agonists. Combined
treatment resulted in peak responses of 0.59 ± 0.06 µmol · h1 · cm
2
(n = 13). We tested the effects of the following
blockers: 5 µM Sch-28080 to probe for the gastric-type
K+-H+-ATPase, 100 µM ouabain to probe
for the non-gastric-type K+-H+-ATPase, 100 nM
bafilomycin A1 to probe for the V-type
H+-ATPase, 400 µM amiloride to probe for the
Na+/H+ exchanger, and 200 µM
ZnCl2 to block H+ channels. All blockers were
added to the mucosal side. Figure 2, D, E, and
G, shows typical blocker experiments, and Fig. 2F summarizes the average effects of blockers. Both bafilomycin
A1 and Sch-28080 are highly specific and selective
blockers. Neither showed significant effects on JH (effects
not different from zero, 1-sample t-tests), indicating that
the V-type H+-ATPase and the gastric-type
K+-H+-ATPase do not significantly contribute to
H+ secretion across airways. Similarly, mucosal ouabain
(Fig. 2, G and F) showed very small effects that were not
significantly different from zero, indicating that the non-gastric-type
K+-H+-ATPase contributes little to
H+ secretion.
Addition of amiloride to the mucosal bath had variable effects on
H+ secretion. In 4 of 11 cultures tested JH was
transiently inhibited by 0.17 ± 0.07 µmol · h
1 · cm
2 (peak
inhibition), corresponding to an inhibition of 34% of total H+ secretion. Figure 2D shows an example. In the
other seven cultures tested amiloride had no significant effect
(0.02 ± 0.03 µmol · h
1 · cm
2). In Fig.
2F the total average effect of amiloride is given. Interestingly, the cultures that showed an amiloride-sensitive JH also expressed a larger amiloride-sensitive
Vt. In these cultures amiloride blocked 76 ± 19% of Vt, whereas in the cultures that did
not show an amiloride-sensitive JH,
Vt was blocked by only 17 ± 8%. This
suggested that the effects of amiloride on JH were likely
not mediated by an apical Na+/H+ exchanger.
However, the data are consistent with the notion that the activity of
apical Na+ channels affected JH (see
DISCUSSION).
ZnCl2 (200 µM) added to the mucosal bath caused sustained block of JH (Fig. 2, E and G). ZnCl2 was the only blocker used that consistently blocked a large fraction of JH. In nine of nine cultures tested, 200 µM ZnCl2 blocked on average 70 ± 8.8% of JH. These data suggest that a zinc-sensitive H+ conductance is the major mechanism for the transepithelial JH in airway cultures.
In addition, we measured H+ secretion in cultures that were
incubated on the mucosal side with nominally ion-free solution (300 mM
mannitol) to determine the ion dependence of the apical mechanism.
Serosal solution was standard NaCl-HEPES Ringer solution. Under these
conditions the basal rate of acidification was 0.07 ± 0.03 µmol · h1 · cm
2
(n = 3). Addition of ATP to the mucosal bath resulted
in an increase of JH of 0.28 ± 0.02 µmol · h
1 · cm
2. An
example is shown in Fig. 2H. Addition of histamine resulted in peak increases of JH of 0.18 ± 0.03 µmol · h
1 · cm
2
(n = 2), and histamine plus ATP resulted in increases
of 0.68 ± 0.08 µmol · h
1 · cm
2
(n = 2). The rates measured in ion-free
solutions were not different from the rates obtained in NaCl Ringer solutions.
The zinc sensitivity and the ion independence of stimulated
H+ secretion indicated an apical H+
conductance. Therefore, JH should be dependent on the
electrochemical driving force across the apical membrane. To test this
hypothesis transepithelial currents were passed across the epithelium.
Airway cultures were bathed in NaCl Ringer, and transepithelial current was clamped for 10 min sequentially to 200, 0, or +200
µA/cm2 before and after ATP stimulation. Figure
3 shows JH measured at
different currents. It should be noted that JH was positive at all clamped currents. Positive currents (which depolarize the apical
membrane) significantly increased JH . The inhibition of JH by negative currents (which hyperpolarize the apical
membrane) was less than the observed stimulation by positive currents.
This relation suggested outward rectification of H+
currents with respect to the apical membrane potential.
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To measure H+ currents directly, single JME airway cells
were whole cell patch-clamped under conditions selective for
H+ currents (see METHODS). For the patch-clamp
experiments JME cells were used in preference to primary ciliated
airway cells because of 1) the difficulty in obtaining
high-resistance seals on ciliated cells and 2) the poor
space clamping of the apical membrane of ciliated cells. H+
currents were measured selectively under conditions adapted from the
measurement of H+ currents in alveolar cells
(3), and cells were prestimulated with 100 µM ATP in the
bath. H+ currents were identified by 1) reversal
potential, 2) voltage-dependent activation, and
3) sensitivity to zinc. Figure
4 shows typical whole cell recordings. In
the presence of a pH 5.3-to-pH 7.3 proton gradient from cell to bath
H+ currents showed a negative reversal potential of
72 ± 6.1 mV (n = 13). When membrane potential
was stepped to depolarizing potentials, currents showed slow activation
and strong outward rectification (Fig. 4, A and
B), which are typical characteristics of H+
currents (3). On average, we found a steady-state
H+ conductance of 9.2 ± 3.8 pS/pF (n = 13, chord conductance at 0 mV, with an average cell capacitance of
29 ± 4 pF). Figure 4C shows a whole cell patch-clamp
experiment in which the effect of ZnCl2 was tested. The
membrane potential was clamped to
80 mV and pulsed to +20 mV every
10 s to continuously monitor the voltage activation of the
H+ current as an identifier. Before addition of
ZnCl2, large currents activated during the depolarizing
pulses. Addition of ZnCl2 to the bath readily blocked the
voltage-activated positive (outward) currents. Figure 4D
shows details of the voltage-activated currents before and after
addition of ZnCl2.
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In vivo the main source for mucosal histamine in the airways is
probably mucosal mast cells. To test the direct effect of mast cells on
H+ secretion by the airway epithelium we used the human
mast cell line HMC-1 (2). HMC-1 cells constitutively
degranulate and release histamine and other factors (2).
HMC-1 cells were suspended in unbuffered NaCl Ringer, the pH was
adjusted to 7.3, and a cell suspension containing 1.5 × 106 HMC-1 cells was added to the mucosal compartment.
Figure 5A shows a typical
experiment. Addition of HMC-1 cells resulted in a large activation of
H+ secretion. On average, JH values of
0.87 ± 0.33 µmol · h1 · cm
2
(n = 3; peak value) were stimulated, which is a
significantly larger response than treatment with histamine (0.27 ± 0.09 µmol · h
1 · cm
2;
Fig. 2C). The increased acidification may have been caused
by another factor released by the mast cells or by H+
release by the mast cells. This latter possibility was tested by adding
HMC-1 cells to experiments without airway cultures (Fig. 5B). When added, 1.5 × 106 HMC-1 cells
showed a brief release of acid with a rate of 0.56 ± 0.028 µmol/h (n = 4 experimental runs). Within <10 min
JH from HMC-1 cells returned to zero, indicating that parts
of the initial peak, but not the continuous acid secretion by airway
epithelium, was caused by a brief acid release from HMC-1 cells.
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We next performed measurements in the presence of 25 mM
HCO22.7 ± 5.4 mV, and
Rt was 730 ± 128
· cm2 (n = 7). The mucosal
medium initially either acidified (5 of 7 cultures) or alkalinized (2 of 7 cultures). On average, basal JH was
0.15 ± 0.28 µmol · h
1 · cm
2 (not
significantly different from zero). When stimulated with both
histamine and ATP, six of seven cultures responded with an increased
rate of acidification of the mucosal medium and one culture showed an
increase in the rate of alkalinization of the mucosal medium. On
average, after stimulation JH was 1.03 ± 0.51 µmol · h
1 · cm
2
(n = 7). Despite the variability in the responses to
stimulation, mucosal ZnCl2 consistently blocked
JH in all tested cultures by
1.2 ± 0.23 µmol · h
1 · cm
2
(n = 5). After treatment with ZnCl2, three
of five treated cultures alkalinized and two acidified the mucosal
medium (on average, JH =
0.51 ± 0.33 µmol · h
1 · cm
2;
n = 5). All average changes in JH measured
in the presence of HCO
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DISCUSSION |
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We report here that human airway surface epithelium shows secretion of H+ into the airway lumen regulated by mucosal histamine or ATP. The effect of histamine suggests that the stimulation of H+ secretion in vivo involves mucosal mast cells, which we also show to elicit acid secretion. Thus from our data we propose that during airway inflammation, such as in asthma, in CF, or during allergic reactions, H+ secretion by the airways is activated and the ASL acidifies. In addition, ATP is released by cells under various conditions including airway epithelium during mechanical stress (18). Therefore, stimulants that are unrelated to the mast cell-mediated responses can also lead to acidification of the ASL.
Properties of channel-mediated H+ secretion in airways. We aimed to identify the type of H+ transporter that operates in the apical cell membrane of airways. We used drug sensitivity and voltage- and ion dependence to characterize the H+ transporter. Our data show that H+ secretion by airways occurred primarily via a zinc-sensitive apical membrane H+ conductance for the following reasons. 1) Under ion-free conditions or in the presence of ions rates of H+ secretion were similar. Therefore, the ion-dependent H+ transporters (i.e., the Na+/H+ exchanger and the K+-H+-ATPase) did not contribute significantly. 2) In transepithelial current-clamp experiments the application of positive (outward) current across the epithelium (which polarizes the cytoplasmic face of the apical membrane positively) was accompanied by an increase in H+ secretion. Current passed in the opposite direction reduced H+ currents, indicating that H+ secretion is electrogenic. 3) In whole cell recordings typical identifiers of H+ currents were found including strong outward rectification and activation by depolarization (3, 9, 25, 35). 4) ZnCl2 effectively blocked H+ secretion in transepithelial recordings and in whole cell patch-clamp recordings. In transepithelial recordings Sch-28080, bafilomycin A1, or ouabain had no significant effects on H+ secretion, indicating that the K+-H+-ATPase and the V-type H+-ATPase did not significantly contribute to transepithelial H+ secretion. Thus the results of the blocker experiments are consistent with the results obtained from the ion-free experiments. An apically localized H+ conductance in human airways was identified by its pharmacological and biophysical characteristics, and other transporters could be excluded by the observed ion independence of H+ secretion and by using blockers. Interestingly, in parallel experiments with bovine tracheal cultures we found a significant block of H+ secretion by bafilomycin A1 (data not shown), indicating species-specific variations in H+ transporters in the airways.
Owing to the strong outward rectification of the H+ currents and the localization of the conductance to the apical membrane, this pathway is expected to support H+ fluxes in the secretory direction. This is consistent with our observation that only H+ secretion (and not H+ absorption) was measured in this report (see, for example, Fig. 3). Our data are consistent with the notion that an outward electrochemical H+ gradient exists across the apical membrane that drives H+ secretion across the airway epithelium. H+ conductances in other cell types have been shown to be extremely dependent on the membrane potential such that at negative potentials currents were very small but at depolarizing and positive potentials H+ currents activated. An additional critical feature is the activation of the H+ conductance by an inside-to-outside H+ gradient, as shown in detail for the H+ conductance in rat alveolar cells (8). In fact, in the absence of an H+ gradient the threshold voltage for activation was shown to be nonphysiologically high, current activation was slow, and the currents were very small (8). Thus, in cell types where H+ channels were found, their assumed function is the dissipation of high intracellular H+ concentrations during metabolic acidosis. For example, in phagocytic neutrophils the intracellular space near the membrane acidifies by release of H+ from NADPH during generation of superoxide anions by the membrane-bound NADPH oxidase, and H+ currents are driven by the metabolic acidosis at the membrane (15). Similarly, a H+ conductance in mast cells has been proposed to dissipate the stimulus-induced cytosolic acidification (25). However, in cultured human nasal airway cells cytosolic pH was reported to be 7.15 (30), which under physiological conditions would result in a H+ gradient from the ASL (pH = 6.9) into the cell. Because our data are consistent with an outward H+ gradient, we suggest that the intracellular pH near the apical membrane is markedly acidic in airway cells. This hypothesis relies on the following observations. Mucosal pH of airway cultures equilibrated at pH = 6.85 (Fig. 1). Furthermore, the apical membrane potential (Va) in human airway cell cultures has been reported asProton permeability in airways.
Our transepithelial measurements were done at a mucosal pH of 7.3, and
peak responses with single agonists were JH = 0.41 µmol · h1 · cm
2 (Fig. 1).
Assuming an intracellular pH of 6.5, Va =
22.5 mV and an amplification of the apical membrane area by the cilia by a factor of 35 (calculated from morphometric data in Ref.
32), then the H+ permeability (P)
of the apical membrane during the peak responses is
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Effects of amiloride on apical Na+ channels vs. Na+/H+ exchanger. Amiloride was used to test for the presence of an apically localized Na+/H+ exchanger. Previously, Paradiso (30) found the Na+/H+ exchanger exclusively in the basolateral membrane. We found that H+ secretion was transiently blocked by amiloride in tissues that also expressed a significant amiloride-sensitive Vt. Variable amiloride-sensitive Na+ currents can be expressed by different cultures, depending in part on the culture conditions, source of cells, and experimental conditions (6, 24, 34). Na+ current across the apical membrane is a significant determinant of Va. Thus the measured inhibition of H+ secretion by amiloride is well explained by an amiloride-induced hyperpolarization of Va.
Role of acidic ASL in asthma.
In asthma, the pH of condensed exhaled breath has been reported to be
markedly acidic (19), a result that could reflect increased rates of acid secretion by the airway epithelium. Asthma is
characterized by an increased number of luminal mast cells (12) that release their contents more readily than normal,
and the levels of mast cell degranulation products in the ASL are correspondingly higher than normal (10). Thus we propose
that increased levels of histamine (and possibly other mast cell
products) in the lumen of the airways of asthmatic patients causes an
acidification of the ASL. At the histamine-stimulated rates of
H+ secretion that we have found (0.41 µmol · h1 · cm
2) and an
estimated buffer capacity of the ASL of 40 mM/pH, the initial rate of
acidification of the ASL can be calculated as 0.17 pH units/min. Acidic
ASL then produces as yet undetermined functional changes in the airway
epithelium that might initiate or exacerbate asthma attacks. Mast cells
1) secrete various factors in addition to histamine
(11) and 2) express H+ channels and
release H+ (25). Therefore, we tested directly
the effect of applying a suspension of the mast cell line HMC-1 to the
mucosal surface of tracheal culture. The resulting H+
secretion by the epithelium was significantly larger than that seen
with histamine or ATP. However, mast cells alone showed a brief but
significant release of acid (Fig. 5), suggesting that the continuously
elevated H+ secretion by the airway cultures was mediated
by mast cell-derived factors.
Role of acid secretion in CF and bacterial colonization.
Asthma and CF share some common symptoms, such as sinusitis and
frequent respiratory infections. CF is caused by a mutation in the CFTR
Cl channel. CFTR is also conductive for
HCO
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ACKNOWLEDGEMENTS |
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We thank Dr. Walter Finkbeiner (University of California, Davis) for providing the airway cultures, Dr. Terry Machen (University of California, Berkeley) for discussion, and Eric Wunderlich for the JME cell cultures.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant 1-P50-HL-60288.
Address for reprint requests and other correspondence: H. Fischer, Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609-1673 (E-mail: hfischer{at}chori.org; http://www.chori.org/scientists/fischer.html).
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.
10.1152/ajpcell.00369.2001
Received 2 August 2001; accepted in final form 8 November 2001.
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