Departments of 1 Molecular and Cellular Physiology, 2 Gastroenterology, and 3 Neurology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932; and 4 First Department of Internal Medicine, Nagoya City University Medical School, Nagoya, Japan
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ABSTRACT |
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It is strongly suspected that
cytokine-induced gene expression in inflammation is oxidant mediated;
however, the intracellular sources of signaling oxidants remain
controversial. In inflammatory bowel disease (IBD) proinflammatory
cytokines, such as TNF-, trigger gene expression of endothelial
adhesion molecules including mucosal addressin cell adhesion molecule-1
(MAdCAM-1). MAdCAM-1 plays an essential role in gut inflammation by
governing the infiltration of leukocytes into the intestine. Several
groups suggest that endothelial-derived reduced NADP (NADPH) oxidase
produces signaling oxidants that control the expression of adhesion
molecules (E-selectin, ICAM-1, VCAM-1). In addition to NADPH oxidase,
cytochrome P-450 (CYP450) monooxygenases have also been
shown to trigger cytokine responses. We found that in high endothelial
venular cells (SVEC4-10), multiple inhibitors of CYP450 monooxygenases
(SKF-525a, ketoconazole, troleandomycin, itraconazole) attenuated
TNF-
induction of MAdCAM-1, whereas NADPH oxidase inhibition (PR-39)
did not. Conversely, E-selectin, ICAM-1, and VCAM-1 induction
requires both NADPH oxidase and CYP450-derived oxidants. We show here
that MAdCAM-1 induction may depend exclusively on CYP450-derived
oxidants, suggesting that CYP450 blockers might represent a possible
novel therapeutic treatment for human IBD.
cytochrome P-450 monooxygenase; reduced nicotinamide adenine dinucleotide phosphate oxygenase; mucosal addressin cell adhesion molecule-1; inflammatory bowel disease; endothelial cell adhesion molecules
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INTRODUCTION |
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INFLAMMATION such as inflammatory bowel disease (IBD) is characterized by the extravasation of leukocytes into the tissues, where these cells induce and sustain chronic intestinal inflammation (19). Leukocyte homing both to normal tissues and to sites of inflammation is regulated in part by differential expression of cell surface homing receptors, such as cell adhesion molecules (CAMs) (7, 23, 31, 33).
Both leukocyte and endothelial CAMs participate in the transmigration of leukocytes from the vascular compartment to tissue sites of inflammation. Endothelial cell adhesion molecules (ECAMs), e.g., E-selectin, ICAM-1, VCAM-1, and mucosal addressin cell adhesion molecule-1 (MAdCAM-1), contribute to the development of chronic inflammation by recruiting leukocytes, especially lymphocytes, into tissues. Tissue infiltration by leukocytes is a common hallmark of inflammation and involves the regulated expression of ECAMs. It is now strongly suspected that ECAM, e.g., E-selectin, ICAM-1, and VCAM-1, expression in response to inflammatory cytokines is oxidant dependent (1, 6, 20, 26, 29, 34), and the precise source of these intracellular signaling oxidants remains unclear.
Both enzymatic and nonenzymic sources of signaling reactive oxygen
species (ROS) have been proposed, including reduced NADP (NADPH)
oxidase, xanthine oxidase, cyclooxygenases, catecholamine oxidation,
and ionizing radiation (17). Several prior studies have
suggested that a phagocyte-type NADPH oxidase, existing within endothelial cells, might be an important source of signal oxidants (1, 12, 14, 22). Cytochrome P-450 (CYP450)
monooxygenases have also recently been proposed as another source of
intracellular signaling ROS (8, 27, 30). Therefore, in the
current study we compared the contributions of endothelial NADPH
oxidase and CYP450 monooxygenase-derived oxidants in TNF--induced
expression of ECAMs.
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MATERIALS AND METHODS |
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Reagents.
N-acetyl-L-cysteine (NAC) and
pyrrolidinedithiocarbamate (PDTC) were purchased from Sigma (St. Louis,
MO). Antibodies to VCAM-1 (clone MK1.9) and ICAM-1 (clone YN1.7.4) were
purchased from Southern Biotechnology Associates (Birmingham, AL), and
antibodies to E-selectin (clone 10Eg.6) and MAdCAM-1 (clone MECA 367)
were purchased from Pharmingen (San Diego, CA). Recombinant mouse
TNF- was purchased from Endogen (Stoughton, MA); fluconazole was
purchased from Pfizer (New York, NY). Troleandomycin and
4-bromophenacylebromide (BPB) were purchased from Sigma. Itraconazole
was purchased from Abbott Laboratories (North Chicago, IL).
Ketoconazole, SKF-525a, and diphenyleneiodonium (DPI) were purchased
from Biomol (Plymouth Meeting, PA) and proline-arginine-rich
antibacterial peptide PR-39 was obtained as a generous gift from Dr.
Chris Ross (Kansas State University, Manhattan, KS).
Isolation of neutrophils. Human neutrophilic polymorphonuclear leukocytes (PMN) were isolated from venous blood of healthy adults with standard dextran sedimentation and gradient separation on Histopaque 1077 (Sigma; Refs. 13, 36). This procedure yields a PMN population that is 95-98% viable (by Trypan blue exclusion) and 98% pure (by acetic acid-crystal violet staining).
Measurement of NADPH oxidase activity. The superoxide anion produced by human PMN NADPH oxidase was determined from the visible absorption spectra, which is based on obtaining the reduction of cytochrome c by superoxide. PMN (2 × 106 /ml) in HBSS with 0.45% (wt/wt) glucose and 0.1% (wt/wt) cytochrome c (Sigma) were pretreated (20 min) with inhibitors (in µM: 20 SKF-525a, 10 ketoconazole, 20 troleandomycin, 1 itraconazole, 10 fluconazole, 0.5 DPI, and 10 PR-39) or superoxide dismutase (SOD; 10 µg/ml) and then cotreated (30 min) with phorbol 12-myristate 13-acetate (PMA; 1 µM). Samples were centrifuged at 7,500 rpm for 5 min, and 200 µl of supernatant were transferred to 96-well plates and read on a Titertek MCC340 plate reader (Titertek Instruments, Huntsville, AL) with absorbance at a wavelength of 450 nm. For experiments, each treatment was performed at least in triplicate.
Chemicals and CYP450 (3A4) interaction. Direct effects of the different inhibitors used in this study on the activity of CYP450 3A4 (CYP3A4) activity was measured with the Vivid CYP3A4 Green Screening Kit (PanVera, Madison, WI) which is a direct fluorometric test for CYP3A4 activity. Wells that did not contain inhibitors but did contain CYP3A4 enzyme plus the fluorescent substrate were used as a "100% activity" control (i.e., no inhibitory effect on CYP3A4 activity). Other wells were identical except for the presence of the particular inhibitor. SKF-525a (20 µM), ketoconazole (10 µM), troleandomycin (20 µM), itraconazole (1 µM), fluconazole (10 µM), DPI (0.5 µM), and PR-39 (10 µM) were tested with the Vivid assay procedure in 96 wells according to the manufacturer's instructions. After all reactions, CYP3A4 activity was read on a Fluoroskan Ascent (Labsystems, Helsinki, Finland) set for excitation at 485 nm and emission at 538 nm. The data are expressed as a percentage of this maximum control fluorescence level. For each experiment, treatments were performed at least in triplicate.
Cell culture. The SVEC4-10 line is an endothelial cell line derived by SV40 (strain 4A) transformation of murine small vessel endothelial cells originally isolated from the axillary lymph node vessels of an adult male C3H/Hej mouse (5). This cell line was maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS) and 1% antibiotic-antimycotic. Cells were seeded into 24-well tissue culture plates at ~20,000 cells/cm2, and experiments were performed immediately on cells reaching confluence.
Western analysis of cell lysates.
Monolayers were either pretreated (1 h) with pharmacological blockers
and then cotreated with TNF- (20 ng/ml; 24h) or not treated with
these test agents and then treated with TNF-
(24 h). All cell
samples were harvested at 24 h. Equal quantities of protein (75 µg) from each of the samples were electrophoretically separated on
7.5% SDS-PAGE gels. Gels were transferred to nitrocellulose membranes
(Sigma) and blocked with 5% milk powder in PBS at 4°C (overnight).
These membranes were washed two times for 10 min with wash buffer
(0.1% milk powder in PBS). Primary rat anti-mouse MAdCAM-1 antibody
was added at a concentration of 10 µg/ml and incubated at room
temperature for 2 h. These membranes were washed twice with wash
buffer. Secondary goat anti-rat horseradish peroxidase-conjugated antibody (Sigma) was added at a 1:2,000 dilution for 2 h. Finally, membranes were washed three times and developed with the enhanced chemiluminescence (ECL) detection system (Amersham, La Jolla, CA). The
density of MAdCAM-1 staining was measured by scanning the 60-kDa band
with an HP ScanJet flatbed scanner. Images were analyzed for density
with Image Pro Plus image analysis software (Media Cybernetics). The
data are expressed as a percentage of TNF-
-induced level of density.
In each protocol, treatments were performed at least in triplicate
(n = 3).
Endothelial cell adhesion expression assay.
The surface expression of ECAMs was measured with the method of
Khan et al. (15). SVEC monolayers were grown in 48-well plates as described by Khan et al. and pretreated (1 h) with inhibitors (in µM: 20 SKF-525a, 10 ketoconazole, 20 troleandomycin, 1 itraconazole, and 10 fluconazole, and 10 PR-39 with 500 nM DPI) and
then cotreated with TNF- (20 ng/ml) at 37°C in medium for 24 h. The cells were washed three times with 0.5 ml of HBSS-PBS
(1:1) at 24 h, and monolayers were incubated with anti-mouse
ICAM-1, anti-VCAM-1, anti-E-selectin, or anti-MAdCAM-1 antibody. All
antibodies were added to cultures after treatment at a concentration of
1 µg/ml in HBSS-PBS + 5% FCS at 37°C for 30 min. Monolayers
were then washed twice with 0.5 ml HBSS-PBS and incubated with
horseradish peroxidase-conjugated rabbit anti-rat IgG (1:2,000
dilution; Sigma) in HBSS-PBS + 5% FCS at 37°C for 30 min.
Monolayers were washed four times with 0.5 ml HBSS-PBS followed by
incubation with 0.25 ml of 0.003% hydrogen peroxide + 0.1 mg/ml
3,3',5,5'-tetramethylbenzidine (Sigma) at 37°C for 60 min in the
dark. The color reaction was stopped by adding 75 µl of 8 N
H2SO4, and the samples were transferred to
96-well plates. Plates were read on a Titertek MCC340 plate reader
(Titertek Instruments) at 450 nm. Blanking (i.e., background) was
performed on monolayers stained only with second antibody. In each
protocol, treatments were performed at least in triplicate (n = 3).
Statistical analysis.
Results are expressed as means ± SE. Significant
differences were assessed by one-way ANOVA plus Fisher's paired least
significant difference (PLSD) test. P values 0.05
were accepted as statistically significant.
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RESULTS |
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TNF--induced MAdCAM-1 expression is oxidant sensitive.
To show the dependence of TNF-
-induced endothelial MAdCAM-1
expression on intracellullar oxidants (as previously shown in the
upregulation of other ECAMs like E-selectin, VCAM-1, and ICAM-1), we
pretreated monolayers with NAC and PDTC, two potent intracellular antioxidants, before TNF-
treatment to block MAdCAM-1 induction. SVEC endothelial cells pretreated with NAC (Fig. 1A; 10 mM) or with PDTC (Fig. 1B; 20 µM) showed significantly reduced TNF-
-induced MAdCAM-1 expression
(measured by Western blot analysis), supporting oxidants as key signals
in TNF-
-induced MAdCAM-1 expression.
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Tests for enzyme inhibitor specificity.
In this study, we wanted to identify the sources of the signaling
oxidants induced by TNF- in the regulation of MAdCAM-1. Because the
studies in Fig. 1 showed a strong dependence of MAdCAM-1 induction on
intracellular oxidants, we next tried to distinguish whether these
effects were due to NADPH oxidase-derived-oxidants or to CYP450
monooxygenase-derived oxidants. Therefore, a panel of highly specific
inhibitors for NADPH oxidase and for CYP450 were selected and their
abilities to block these enzymes were analyzed by two assays for these
respective systems: neutrophil superoxide formation (NADPH oxidase) and
a direct assay for CYP3A4 monooxygenase.
Effect of blockers used in this study on neutrophil NADPH oxidase.
To screen the drugs used in this study for their ability to block
NADPH oxidase-dependent superoxide production, human PMN were used as a
model NADPH oxidase-dependent superoxide-generating system, because PMN
will form superoxide by the action of NADPH oxidase. Superoxide
production and inhibition in this experiment were measured
spectrophotometrically by the reduction of cytochrome c. PMA
(1 µM; n = 6)-stimulated superoxide formation was
considered to be "100%" or maximal activation. The production of
superoxide by PMN was prevented by superoxide dismutase (10 µg/ml;
n = 6), which demonstrates the specificity of this
assay for the superoxide anion. DPI (0.5 µM; n = 4),
a nonselective inhibitor for NADPH oxidase, and PR-39 (10 µM;
n = 4), a specific NADPH oxidase blocker, almost
completely inhibited NADPH oxidase-dependent superoxide formation.
Troleandomycin (20 µM; n = 6) and fluconazole (10 µM; n = 6) slightly increased superoxide formation
over PMA-stimulated levels. In contrast, ketoconazole (10 µM;
n = 6), SKF-525a (20 µM; n = 6), and
itraconazole (1 µM; n = 6) slightly decreased superoxide formation (Fig. 2).
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Effect of CYP450 monooxygenase blockers.
To screen the drugs used in this study for their ability to
directly inhibit CYP450 monooxygenase, CYP3A4 activity was measured with the Vivid CYP3A4 Green screening kit. SKF-525a (20 µM;
n = 5), ketoconazole (10 µM; n = 5),
troleandomycin (20 µM; n = 5), itraconazole (1 µM;
n = 5), and DPI (0.5 µM; n = 5) all
strongly inhibited CYP3A4 activity. Fluconazole (10 µM;
n = 5), a CYP2C9 inhibitor, only slightly reduced
CYP3A4 activity, whereas PR-39 (10 µM; n = 4), a
selective NADPH oxidase inhibitor, slightly increased CYP3A4 activity
(Fig. 3).
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Effect of CYP450 and NADPH oxidase antagonists on MAdCAM-1
expression.
To quantitate which oxidant contributes the most in the
expression of MAdCAM-1 on SVEC endothelial cells, CYP450 and/or NADPH oxidase inhibitors were preincubated and coincubated with these blockers in the presence or absence of TNF-. CYP450 inhibitors [SKF-525a (20 µM; n = 4), ketoconazole (10 µM;
n = 3), troleandomycin (20 µM; n = 3), and itraconazole (1 µM; n = 3), proven to have specific inhibition against CYP3A4 activity] and DPI (0.5 µM; n = 3), a nonselective blocker of multiple CYP450
isoforms and NADPH oxidase, significantly decreased TNF-
-induced
MAdCAM-1 expression. Fluconazole (10 µM; n = 3), a
CYP2C9 blocker, and the NADPH oxidase inhibitor PR-39 (10 µM;
n = 3) did not alter MAdCAM-1 expression by Western
blotting. In addition, the phospholipase A2 inhibitor BPB
(10 µM; n = 3) prevented the TNF-
-induced
expression of MAdCAM-1 (Fig. 4).
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Effect of blockers on expression of E-selectin, ICAM-1, and VCAM-1.
To evaluate which oxidase is most important in the expression of
other ECAMs CYP450 and/or NADPH oxidase inhibitors were incubated with
SVEC in the presence (and absence) of TNF-. Both a CYP450 inhibitor
(SKF-525a, 20 µM; n = 8) and a NADPH oxidase
inhibitor (PR-39, 10 µM; n = 8) prevented E-selectin
expression assessed via surface expression assay (Fig.
5A). Both CYP450 and NADPH oxidase inhibition also prevented the TNF-
-induced expression of
ICAM-1 (Fig. 5B). Similarly, both CYP450 and NADPH oxidase inhibition blocked the expression of VCAM-1 (Fig. 5C).
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DISCUSSION |
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Cytokine-dependent leukocyte-mediated tissue injury is induced in large part by the adhesion and extravasation of several classes of leukocytes that infiltrate inflamed tissues. The motility of these leukocytes depends on the increased expression of several endothelial adhesion molecules whose synthesis depends on the formation of signaling oxidants formed in response to inflammatory cytokines. The expression of these molecules and tissue inflammatory injury appears to be linked to oxidants (16), because this type of injury is largely reversed by treatment with several antioxidants (11, 18, 25).
Regulating the hyperexpression of ECAMs associated with many chronic
inflammatory states is now recognized as one of the most effective,
novel approaches for limiting leukocyte-mediated tissue damage.
Although it is well accepted that the expression of ECAMs (E-selectin,
ICAM-1, and VCAM-1) in response to inflammatory cytokines is oxidant
dependent (6, 20, 26, 28, 29, 34), there are a few reports
that claim that oxidants may not be required for the activation of
NF-B, the major transcription factor activated by cytokines in
inflammation (3) (2). Therefore, the first goal of this study was to confirm that TNF-
-induced MAdCAM-1 expression is, in fact, oxidant dependent. We showed that two different
intracellular antioxidants, NAC and PDTC, each prevent TNF-
-induced
MAdCAM-1 expression, suggesting for the first time that the regulation
of MAdCAM-1 is oxidant mediated. These data cumulatively suggest that
antioxidants and antioxidant source drugs are capable of downregulating
ECAM expression and consequently blocking the hypermigration of
leukocytes into tissues. On the basis of the effects of PDTC on
intracellular glutathione, some investigators have reported that PDTC
has prooxidant properties (4, 21). It is worthwhile noting
that when PDTC was reported in these studies as a prooxidant, it was
used at high (>25 µM) concentrations. It may also be worth noting
that in those studies where PDTC was a prooxidant, it was used in
nonendothelial cells (EL4.NOB thymoma cells and E6.1 lymphoma cells).
In the current study, we used PDTC at 20 µM and only in SVEC
endothelial cells.
One of the major sources of endothelium-derived signaling oxidants
appears to be an NADPH oxidase similar to that previously reported
within PMN (10, 32). In endothelial cells, NADPH oxidase
activity has also been implicated as a source of signaling oxidants
that trigger NF-B and AP-1 activation under ischemic conditions (35). However, CYP450 enzymes, which have now
been identified as potential "endothelium-derived hyperpolarizing
factor" (EDHF) synthases, have also recently been demonstrated to
generate ROS and might therefore also function as oxidant signal
generators in response to stimuli (9).
In this report, we show that CYP450-derived oxidant signaling plays a significant role in the expression of several ECAMs. Using selective inhibitors of CYP450 and NADPH oxidase, we show here that CYP450 is an important source of ROS-dependent signals induced by cytokines. The effects of NADPH oxidase inhibitors and CYP450 inhibitors on the activity of these systems support the stated pharmacological specificity of the blockers used here. Our findings support the idea that PR-39 is a specific NADPH oxidase inhibitor and that SKF-525a, ketoconazole, troleandomycin, and itraconazole are specific blockers of various CYP450 isoforms.
Interestingly, the CYP450 blockers that displayed inhibition specific
for CYP3A4, were most effective at blocking TNF--induced MAdCAM-1
expression, whereas the specific NADPH oxidase inhibitor (PR-39) did
not. These results are consistent with CYP450-derived ROS, not
NADPH-derived oxidants, as essential intermediates in TNF-
-stimulated expression of MAdCAM-1. With respect to cytokine signaling, our data corroborate and extend findings by Pietersma et al.
(27), who stated that NADPH oxidase-derived ROS do not appear to contribute to cytokine induction of adhesion molecules in
human umbilical vein endothelial cells (HUVEC). Rather, they suggested
that cytokine-induced ECAM expression was due to oxidants formed by
CYP450 activity. In that study, the authors were not able to implicate
a specific CYP450 isoform in the activation of ECAM expression. It is
also worthwhile noting that the opinion of this group was based on
their presumption that HUVEC lacked most components of the
phagocyte-type NADPH oxidase. However, because we find that PR-39
appears to block VCAM-1 expression, our data probably support both
oxidant systems within HUVEC (data not shown). Because fluconazole,
which is reported to be an inhibitor of CYP2C9, had no effect on CYP3A4
activity and also failed to prevent MAdCAM-1 expression, our data
support the CYP3A4 isoform as a key cytochrome in cytokine signaling.
We also examined oxidant dependence in the induction of other ECAMs, (E-selectin, ICAM-1, and VCAM-1) using cell surface expression assays (ELISA). The expression of these three ECAMs appears to require the participation of both NADPH oxidase and CYP450 monooxygenase-derived ROS, because each class of inhibitor was able to reduce the expression of these molecules in response to cytokines.
Although oxidants are excellent candidate metabolites produced by
cytochrome, several prostanoids are also produced by CYP450, including
epoxyeicosatetraenoic acid (EET) and hydroxyeicosatetraenoic acid
(HETE). The prostanoid metabolic pathways are also thought to represent
a source of EDHF. Because these substances are metabolites of
arachidonic acid, it is possible that they might also modulate cytokine-induced ECAM expression in addition to oxidants. However, when
these agents were tested for their effects on ECAM expression, they
were found not to increase, but rather to decrease, the expression of
endothelial adhesion molecules (24). Paradoxically, we
found that a specific phospholipase A2 inhibitor, BPB,
abolished TNF- induction of MAdCAM-1. Our current interpretation of
this result is that CYP450 induction of MAdCAM-1 may require
arachidonic acid as a substrate, which is important in the generation
of oxidant signals, but the final prostanoids derived from these
pathways do not promote ECAM expression.
In summary, ECAM expression in response to cytokines is complex and appears to require oxidants derived from several sources, including NADPH oxidase and CYP450. It is interesting that MAdCAM-1 regulation may be unique in its apparently strict requirement for oxidants derived from CYP450 monooxygenase. The exact reason(s) that MAdCAM-1 expression might show this type of specificity is not currently clear but could reflect interactions between prostanoids and oxidants in the activation of transcription factors, differential sensitivities of transcription factors to oxidants, or as yet unidentified posttranscriptional events. Importantly, the identification of CYP450 as an important oxidant signal generator in inflammation suggests that the large variety of safe and well-tolerated CYP450 antagonists may be effective, novel treatments for many forms of chronic inflammation mediated by cytokines, including arthritis, vasculitis, and some forms of IBD.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43785 and the Biomedical Research Foundation of NW Louisiana.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. S. Alexander, Molecular and Cellular Physiology, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA, 71130-3932 (E-mail: jalexa{at}lsuhsc.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.
First published September 4, 2002;10.1152/ajpcell.00271.2002
Received 14 June 2002; accepted in final form 19 August 2002.
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