Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California School of Medicine, San Diego, California 92103-8382
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ABSTRACT |
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Intracellular K+ plays an important role in controlling the cytoplasmic ion homeostasis for maintaining cell volume and inhibiting apoptotic enzymes in the cytosol and nucleus. Cytoplasmic K+ concentration is mainly regulated by K+ uptake via Na+-K+-ATPase and K+ efflux through K+ channels in the plasma membrane. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore that dissipates the H+ gradient across the inner membrane of mitochondria, induces apoptosis in many cell types. In rat and human pulmonary artery smooth muscle cells (PASMC), FCCP opened the large-conductance, voltage- and Ca2+-sensitive K+ (maxi-K) channels, increased K+ currents through maxi-K channels [IK(Ca)], and induced apoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM) decreased IK(Ca) by blocking the sarcolemmal maxi-K channels and inhibited the FCCP-induced apoptosis in PASMC cultured in media containing serum and growth factors. Furthermore, inhibition of K+ efflux by raising extracellular K+ concentration from 5 to 40 mM also attenuated PASMC apoptosis induced by FCCP and the K+ ionophore valinomycin. These results suggest that FCCP-mediated apoptosis in PASMC is partially due to an increase of maxi-K channel activity. The resultant K+ loss through opened maxi-K channels may serve as a trigger for cell shrinkage and caspase activation, which are major characteristics of apoptosis in pulmonary vascular smooth muscle cells.
mitochondrial membrane potential; cytoplasmic calcium; pulmonary artery smooth muscle cells
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INTRODUCTION |
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PULMONARY ARTERIES have a trilamellar structure that is composed of fibroblasts (adventitia), smooth muscle cells (media), and endothelial cells (intima). In pulmonary artery smooth muscle cells (PASMC), there is a natural balance between proliferation and apoptosis under normal conditions (8, 46). Augmentation of proliferation and inhibition of apoptosis in PASMC would lead to pulmonary medial thickening, which is an early vascular lesion in patients with primary pulmonary hypertension (8, 43, 46, 55). Therefore, an imbalance between PASMC proliferation and apoptosis may play a critical role in the development of pulmonary vascular remodeling. Inhibition of PASMC growth and augmentation of cell apoptosis could also serve as therapeutic approaches for patients with pulmonary hypertension (8, 37, 46, 57).
Cytoplasmic K+ in excitable and nonexcitable cells plays an important role in maintaining intracellular ion homeostasis to control cell volume (4), regulating cell cycle (7, 9), and inhibiting apoptotic enzymes in the cytosol and nucleus (24). Cytoplasmic K+ concentration ([K+]c) is mainly regulated by the activity of Na+-K+-ATPase and various K+ channels in the plasma membrane. The loss of cytoplasmic K+ due to increased K+ efflux through plasmalemmal K+ channels results in cell shrinkage, a major characteristic of apoptosis (4), and caspase activation, a triggering process in apoptosis (24, 45, 52, 56).
The large-conductance, voltage- and Ca2+-sensitive K+ (maxi-K) channels are highly expressed in vascular smooth muscle cells and synergistically regulated by cytoplasmic Ca2+ concentration ([Ca2+]c) and plasma membrane potential (Em) (5, 38, 51, 53, 63). A localized increase in [Ca2+]c (e.g., Ca2+ sparks) in vascular smooth muscle cells, due to Ca2+ release from intracellular Ca2+ stores, opens maxi-K channels, increases K+ currents [IK(Ca)] through maxi-K channels, hyperpolarizes the cell membrane, and causes vasodilation (27, 41). Opening of maxi-K channels would also promote K+ efflux and decrease [K+]c, as a result of their large conductances (200-250 pS), and induce apoptosis (60, 61).
Many metabolic inhibitors increase maxi-K channel activity by releasing
Ca2+ from intracellular organelles [e.g., mitochondria and
sarcoplasmic/endoplasmic reticulum (S/ER)] (11, 12, 39)
and thus induce cell death. Carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (FCCP), which uncouples
mitochondrial oxidative phosphorylation and inhibits ATP synthesis, is
a protonophore that dissipates the proton gradient across the inner
membrane of mitochondria (19, 23). The H+
gradient is required for maintaining a transmembrane potential in
mitochondria (m), stimulating Ca2+
accumulation in mitochondria (19), and causing oxidative
ATP synthesis (23). Therefore, FCCP causes an abolition
(i.e., depolarization) of
m, which subsequently
mobilizes Ca2+ from mitochondria into the cytosol
(11, 12). FCCP induces apoptosis in many cell
types (10). In this study, we used patch-clamp techniques
and digital imaging fluorescence microscopy to test the hypothesis that
FCCP-mediated activation of maxi-K channels contributes to induction of
apoptosis in human and animal PASMC.
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METHODS |
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Cell preparation. Rat PASMC were prepared from pulmonary arteries of Sprague-Dawley rats (150-200 g) (62, 63). The isolated pulmonary arteries were incubated for 20 min in Hanks' balanced salt solution containing 1.5 mg/ml collagenase (Worthington). Adventitia and endothelium were carefully removed after the incubation. The remaining smooth muscles were then digested with 1.5 mg/ml collagenase and 0.5 mg/ml elastase (Sigma Chemical) at 37°C. The cells were plated onto 25-mm coverslips and incubated in DMEM containing 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 in air at 37°C. Human PASMC (Clonetics) were seeded in flasks at a density of 2,500-3,500 cells/cm2 and incubated in smooth muscle growth medium (Clonetics). The medium was changed after 24 h and every 48 h thereafter. Smooth muscle growth medium is composed of smooth muscle basal medium, 5% FBS, 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor, and 5 µg/ml insulin. Cells were subcultured or plated onto 25-mm coverslips using trypsin-EDTA buffer (Clonetics) when 70-90% confluence was achieved. The cells at passages 4-6 were used for experimentation.
Electrophysiological measurement.
Whole cell and single-channel K+ currents
(IK) were recorded with an Axopatch-1D amplifier
and a DigiData 1200 interface (Axon Instruments) using patch-clamp
techniques (20, 62). Patch pipettes (2-4 M) were
fabricated on a Sutter electrode puller using borosilicate glass tubes
and fire-polished on a Narishige microforge. Command voltage protocols
and data acquisition were performed using pCLAMP software (Axon
Instruments). Currents were filtered at 1-2 kHz (
3 dB) and
digitized at 2-4 kHz using the amplifier. In experiments with
cell-attached patches, a gigaohm seal was achieved using fire-polished
glass electrodes filled with a high-K+ (135 mM) solution.
The bath solution was the standard physiological salt solution (PSS)
with 4.7 mM KCl. Under these conditions, the actual patch membrane
potential was unknown; however, it was assumed that the patch membrane
potential is equal to the difference between the pipette command
potential and the actual resting membrane potential (which is about
40 mV in the cell preparation used in this study) (62,
63). Thus voltages are expressed as pipette (or applied command)
potentials. All experiments were performed at room temperature
(22-24°C).
Immunocytochemistry. The cells, grown on 10-mm coverslips, were first washed with PBS (Sigma Chemical) and then fixed in 95% ethanol and stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (DAPI; Sigma Chemical). DAPI (5 µM) was dissolved in an antibody buffer containing 500 mM NaCl, 20 µM NaN3, 10 µM MgCl2, and 20 µM Tris-HCl (pH 7.4). The blue fluorescence emitted at 461 nm was used to visualize the cell nuclei. The DAPI-stained cells were examined with a fluorescence microscope (model TE 300, Nikon), and the cell (nuclear) images were acquired using a high-resolution fluorescence imaging system (Solamere).
For each coverslip, 5-10 fields (~20-25 cells/field) were randomly selected to determine the percentage of apoptotic cells in total cells on the basis of the morphological characteristics of apoptosis: cell (nuclear) shrinkage, nuclear condensation, and nuclear breakage. The cells with clearly defined nuclear breakage, remarkably condensed nuclear fluorescence, and significantly shrunken cell body and nucleus were defined as apoptotic cells. The relative cross-sectional nuclear area of the DAPI-stained cells (on the basis of the area of pixels) was measured using the NIH Imaging software. To quantify apoptosis, TdT-mediated dUTP nick end labeling assays were also performed with the In Situ Cell Death Detection Kit (TMR Red, Boehringer Mannheim); the nuclear morphology was examined by labeling with DAPI.Measurement of rhodamine fluorescence.
The cells, grown on 25-mm coverslips, were loaded with rhodamine 123 (R123, Molecular Probes) by incubation with 10 µg/ml for 30 min at
37°C (11, 12). R123 is taken up selectively by
mitochondria (29, 30), and its uptake is dependent on
m. R123 fluorescence was excited at 488 nm and
measured at 530 nm using a GEN IV charge-coupled device camera
connected to a microscope (model TE 300, Nikon). In isolated
mitochondria, the relationship between R123 fluorescence and
m is linear (13). The R123 fluorescence, which is quenched at resting
m, increases with
mitochondrial membrane depolarization (11, 12). The R123
fluorescence signals were stored in a Macintosh computer and analyzed
using QVD software (Solamere). The percent change of the R123
fluorescence from the baseline level is used for comparison between responses.
Measurement of [Ca2+]c. The cells were loaded with fura 2-AM (3 µM) for 30 min at 24°C under an atmosphere of 5% CO2-95% air. The fura 2-loaded cells were then superfused with PSS for 20 min at 32°C to wash away extracellular fura 2-AM and to allow sufficient time for intracellular esterases to cleave cytosolic fura 2-AM into the active fura 2. Fura 2 fluorescence (510-nm emission, 360- and 380-nm excitation) from the cells and background was measured using a charge- coupled device camera connected to a Nikon microscope. The fluorescence signals were collected continuously and stored in an IBM-compatible computer for later analysis. The 360- to 380-nm excitation ratios of the fluorescence images were then calculated and calibrated to express [Ca2+]c (18, 62).
Reagents and solutions. For measuring whole cell IK and [Ca2+]c, a coverslip containing the cells was positioned in a recording chamber (~0.75 ml) and superfused (2-3 ml/min) with the standard extracellular (bath) PSS. The PSS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4). In Ca2+-free PSS, CaCl2 was replaced by equimolar MgCl2 and 1 mM EGTA was added to chelate residual Ca2+. The pipette (internal) solution for recording whole cell IK contained (in mM) 135 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 Na2ATP (pH 7.2). For single-channel IK recording in cell-attached patches, the pipette (external) solution contained (in mM) 135 KCl, 4 MgCl2, 10 HEPES, and 10 EGTA (pH 7.4).
FCCP (Sigma Chemical) and valinomycin (Sigma Chemical) were prepared as 20 mM stock solutions in DMSO. Aliquots of the stock solutions were diluted 1:1,000-4,000 into PSS (for electrophysiological and fluorescent experiments) or 10% FBS-DMEM (for immunocytochemical experiments). Similar dilutions of DMSO (0.017-0.05%), alone, were used as vehicle control in PSS or the culture media. Tetraethylammonium (TEA; Sigma Chemical) and iberiotoxin (IBTX; Sigma Chemical) were directly dissolved into PSS or culture media on the day of use. The pH values of all solutions were checked after addition of the drugs and readjusted to 7.4. In high-K+ (25 or 40 mM) solution or culture medium, NaCl in PSS and in the customized DMEM (MediaTech) was replaced, mole-for-mole, by KCl to maintain the solution's osmolarity.Statistics. The composite data are expressed as means ± SE. Statistical analysis was performed using paired or unpaired Student's t-test or ANOVA and post hoc tests (Student Newman-Keuls) where appropriate. Differences were considered to be significant when P < 0.05.
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RESULTS |
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Effects of FCCP on R123 fluorescence
in PASMC.
m is primarily generated by a proton gradient across
the mitochondrial inner membrane (3, 19, 23). Changes in
m were determined in human PASMC loaded with R123
(11, 12); mitochondrial depolarization increases R123
fluorescence. In human PASMC, FCCP significantly increased R123
fluorescence (i.e., depolarized
m; Fig.
1A). Increasing extracellular
K+ concentration from 5 to 25 mM, which decreases the
driving force for K+ efflux, and extracellular application
of 1 mM TEA or 100 nM IBTX, which blocks maxi-K channels, negligibly
affected the R123 fluorescence (Fig. 1). Furthermore, pretreatment of
the cells with 25 mM K+, 1 mM TEA, or 100 nM IBTX had
little effect on the FCCP-induced increases in R123 fluorescence (Fig.
1B). These results indicate that inhibition of
K+ efflux across the plasma membrane, as a result of
reduced K+ driving force or blocked maxi-K channels, does
not interfere with the depolarizing effect of FCCP on
m in PASMC.
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FCCP increases the large-conductance
IK(Ca) in rat PASMC.
A large-amplitude single-channel IK was observed
in cell-attached membrane patches (with symmetrical K+
gradient) of rat PASMC during sustained depolarization to positive potentials (Fig. 2A). Slope
conductance of the channels responsible for the current, determined by
current-voltage relationships obtained from 12 cells, ranged from 200 to 225 pS (218 ± 8 pS). This is consistent with the slope
conductance (200-250 pS) of the large-conductance maxi-K channels
that have been identified and characterized in vascular smooth muscle
cells (5, 41, 42, 44, 53). Thus this large-amplitude
IK in rat PASMC was actually
IK(Ca) resulting from K+ efflux
through the large-conductance maxi-K channels.
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FCCP increases whole cell
IK(Ca) in human PASMC.
Extracellular application of IBTX (100 nM) and TEA (1 mM), blockers of
maxi-K channels (2, 42), significantly decreased whole
cell IK in human PASMC (Fig.
4A, a and
b). Consistent with the single-channel results in rat PASMC
(Figs. 2 and 3A), application of 5 µM FCCP reversibly
increased whole cell IK in human PASMC (Fig.
4Ac). The IBTX-sensitive, TEA-sensitive, and FCCP-activated components of whole cell IK were activated at
approximately 40 mV (Fig. 4B) and show marked outward
rectification at potentials more positive than +40 mV (Fig.
4C). The kinetics of the IBTX-sensitive, TEA-sensitive, and
FCCP-activated components of whole cell IK are
very similar to those of the noisy IK(Ca)
observed in vascular smooth muscle cells (2, 17).
Furthermore, FCCP rapidly decreased membrane input resistance at a
holding potential of 0 mV; only K+ channels were active
under these conditions (the calculated equilibrium potentials for
K+, Cl
, Na+, and Ca2+
were
84,
1.5, +66, and +122 mV, respectively).
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Inhibitory effect of IBTX on
FCCP-induced increase in
IK(Ca) in human PASMC.
In the absence of IBTX in the pipette solution, extracellular
application of 5 µM FCCP significantly increased the activity of
maxi-K channels in cell-attached membrane patches;
NPo was increased 67-fold (from 0.00234 to
0.15999; Fig. 5A). Inclusion of 100 nM IBTX in the pipette solution significantly decreased the
activity of maxi-K channels; averaged NPo values
were 0.0257 ± 0.0212 and 0.00044 ± 0.00006 in the absence
and presence of IBTX, respectively. Furthermore, the FCCP-induced
increase in single-channel IK(Ca) was almost
abolished when 100 nM IBTX was included in the pipette solution (Fig.
5B). These results suggest that the FCCP-induced increase in
IK was mainly due to activation of the
IBTX-sensitive maxi-K channels.
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Inhibitory effect of 40 mM
K+ or TEA
on FCCP-induced apoptosis in
PASMC.
Treatment of rat or human PASMC with FCCP (5-15 µM for 20 h) induced cell (nuclear) shrinkage, nuclear condensation, nuclear breakage, and apoptotic bodies in 15-40% of the cells, while
<3% of the untreated control cells showed these apoptotic
characteristics (Fig. 6, A and
B). Increasing extracellular [K+] from 5 to 40 mM, which attenuates IK by reducing the
K+ driving force, decreased the FCCP-induced
apoptosis by ~30% in rat PASMC (from 32 ± 5 to 22 ± 5%, P < 0.001) and ~47% in human PASMC (from
38 ± 6 to 18 ± 5%, P < 0.001; Fig.
6C). Furthermore, treatment of the cells with 1 mM TEA or
100 nM IBTX, which blocks maxi-K channels, also significantly inhibited
the FCCP-induced apoptosis in rat and human PASMC (Fig.
6C).
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Inhibitory effect of 40 mM
K+ on valinomycin-induced
apoptosis.
The transmembrane K+ efflux is determined by the
K+ electrochemical gradient (driving force) and the
K+ permeability. Valinomycin is a K+ ionophore
that increases K+ efflux and induces apoptosis in
variety of cell types (16, 25), including rat and human
PASMC (Fig. 7A). Increasing
extracellular K+ from 5 to 40 mM, which decreases the
K+ electrochemical gradient, significantly inhibited the
valinomycin-induced apoptosis in rat (from 88 to 63%,
P < 0.001) and human (from 81 to 63%,
P < 0.001) PASMC (Fig. 7B). These results
suggest that FCCP- and valinomycin-induced apoptosis in PASMC
is related to increased K+ efflux, which is caused by
FCCP-activated K+ channels and valinomycin-formed
K+ pores in the plasma membrane.
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DISCUSSION |
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FCCP induced apoptosis with characteristic cell shrinkage,
nuclear condensation, and breakage in PASMC. In rat and human PASMC, FCCP depolarized m, mobilized Ca2+ from
the mitochondria to the cytosol, activated maxi-K channels, increased
IK(Ca), and induced apoptosis. Blockade
of the maxi-K channels by IBTX and TEA or decrease of K+
efflux by reducing the K+ driving force significantly
inhibited the FCCP-induced PASMC apoptosis. These results
suggest that FCCP-mediated apoptosis in PASMC is partially due
to activation of maxi-K channels in the plasma membrane. The resultant
K+ loss through opened K+ channels may be a
trigger for apoptosis in pulmonary vascular smooth muscle cells
(Fig. 8).
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Involvement of K+ efflux through sarcolemmal K+ channels in apoptosis. K+ is the predominant cation in the cytosol. Maintenance of a high [K+] in the cytoplasm (140-150 mM) is essential for 1) governing cell excitability (42), 2) setting resting Em (42), 3) regulating apoptotic enzyme activity (24), and 4) controlling cell volume (4). Cytoplasmic K+ at normal concentration (~140 mM) decreases apoptotic DNA fragmentation and caspase-3-like protease activation (24). Decrease in [K+]c, due to elevated K+ efflux through opened K+ channels, results in cell shrinkage (4, 16, 25) and reduces the inhibitory effect of cytoplasmic K+ on caspase-3-like protease and the internucleosomal DNA cleavage nuclease (24). Caspases and nucleases are major inducers of apoptosis (52). Therefore, an increase in K+ efflux, partially due to activated sarcolemmal K+ channels, is necessary for the initiation of apoptosis (24, 60, 61). The observations in PASMC from the present study are consistent with the results observed in neurons and lymphocytes (24, 60, 61): blockade of K+ channels by TEA or decreasing the K+ driving force by raising extracellular K+ significantly attenuated the apoptosis.
It is unknown whether the apoptosis induced by increasing K+ channel activity depends on the time course of K+ efflux. A transient (or short-term) increase in K+ efflux or cytosolic K+ loss would relieve its tonic suppression on caspase activity and thus trigger the caspase-mediated apoptosis (e.g., in the presence of apoptosis inducers). Because apoptosis is an irreversible process, apoptosis may occur any time when K+ efflux is increased. In in vivo experiments, PASMC apoptosis has been observed in hypertrophied pulmonary arteries (8). Apoptosis can take place in different cell cycle phases; therefore, increasing K+ efflux should be able to work on an already modified pulmonary vascular wall. However, whether apoptosis induced by increased K+ efflux only occurs in the cells that contribute to hypertrophy, but not in the normally controlled cells, is unknown. Further study is needed to define whether apoptosis induced by increasing K+ efflux depends on cell phenotype.Activation of maxi-K channels by FCCP-induced
Ca2+ release.
In vascular smooth muscle cells including PASMC, the large-conductance
maxi-K channels are regulated by cytoplasmic Ca2+ and
Em (5, 42, 53). A localized rise in
[Ca2+]c, due to metabolic inhibition-mediated
Ca2+ mobilization from mitochondria and the S/ER, activates
maxi-K channels and increases IK(Ca) (39,
41, 63). FCCP is a proton ionophore that 1)
depolarizes m by dissipating the H+
gradient across the inner membrane of mitochondria (11, 12, 19), 2) releases Ca2+ from the
mitochondria into the cytosol (11, 12, 19, 36, 64), and
3) inhibits ATP production by uncoupling oxidative phosphorylation (23). There are numerous close contacts
between the mitochondria and S/ER (47), suggesting that
these two organelles may coordinate with each other in releasing
Ca2+ to or sequestering Ca2+ from the cytosol.
Other possible mechanisms involved in apoptosis mediated by
FCCP or K+
channel activation.
Mitochondrial intermembrane space contains several proteins that are
liberated through the outer membrane to participate in initiation of
apoptosis (35, 50). Release of cytochrome
c to the cytosol (31, 59) and translocation of
the apoptosis-inducing factor to the nucleus (50)
initiate the apoptotic cascade. A direct relationship between
m depolarization and the release of cytochrome
c (and apoptosis-inducing factors) has been
demonstrated to play an important role in apoptosis (10,
22, 54). However, whether
m depolarization is
required for apoptosis is still unclear (14, 32).
Summary and conclusion.
The results from this study suggest that FCCP-induced apoptosis
in rat and human PASMC is partially due to activation of maxi-K channels in the plasma membrane. FCCP depolarizes m
and releases Ca2+ from mitochondria to the cytosol. The
local rise in [Ca2+]c activates maxi-K
channels and increases IK(Ca). The resultant K+ loss due to elevated K+ efflux may play an
important role in the onset of apoptosis in pulmonary vascular
smooth muscle cells (Fig. 8). Activation of maxi-K channels by
Ca2+ sparks due to Ca2+ release from
intracellular organelles also triggers vasodilation. Thus development
of drugs directed at activation of K+ channels in PASMC
would be potentially a useful therapeutic approach for treatment of
pulmonary hypertension that is characterized by sustained
vasoconstriction and excessive vascular medial hypertrophy.
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ACKNOWLEDGEMENTS |
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We thank S. S. McDaniel, Y. Yu, and Y. Zhao for technical assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-54043 and HL-64945 (to J. X.-J. Yuan). S. Krick is an Ambassadorial Scholar of the Rotary International. J. X.-J. Yuan is an Established Investigator of the American Heart Association (Grant 974009N).
Address for reprint requests and other correspondence: J. X.-J. Yuan, Div. of Pulmonary and Critical Care Medicine, UCSD Medical Center, 200 W. Arbor Dr., San Diego, CA 92103-8382 (E-mail: xiyuan{at}ucsd.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 23 June 2000; accepted in final form 26 October 2000.
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