Department of Pathology, The University of Western Ontario, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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Human endothelial
cells cultured under high glucose (HG) conditions were shown before to
upregulate several basement membrane proteins, including fibronectin
(FN), thus mimicking effects of diabetes. Using human macrovascular
(HUVEC) and microvascular (HMEC) endothelial cell lines, we evaluated
in the present study some of the key molecular signaling events
involved in HG-induced FN overexpression. This expression was shown to
be dependent on endogenous endothelin (ET) receptor-mediated signaling.
We also examined the roles played by protein kinase C (PKC) and the
transcription factors nuclear factor B (NF-
B) and activating
protein (AP)-1 with respect to such changes. HG, PKC activators, and
ETs (ET-1 and ET-3) that increased FN expression also caused activation of NF-
B and AP-1. Inhibitors of both NF-
B and AP-1 prevented HG-
and ET-induced FN production. ET receptor blockade also prevented these
HG- and ET-mediated changes. The results of this study indicate that
glucose-induced increased FN production in diabetes may be mediated via ET-dependent NF-
B and AP-1 activation.
glucose; endothelial cells; activating protein-1; nuclear
factor-B
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INTRODUCTION |
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INCREASED EXTRACELLULAR
MATRIX (ECM) protein synthesis and capillary basement membrane
(BM) thickening are characteristic features of diabetic
microangiopathy, which is the most common pathological finding in
several chronic diabetic complications, including retinopathy and
nephropathy (18, 4). Various pathogenetic mechanisms may
be responsible for capillary BM thickening in diabetes. Fibronectin
(FN) is one of the most important ECM proteins that mediate a number of
functions in BMs. Physiologically, FN plays an important role in cell
adhesion, motility, tissue repair, etc. However, its overproduction may
decrease motility and replication of many cells, including endothelial
cells (ECs) (24). Diabetes increases turnover of vascular
ECs in the retina (26). Furthermore, FN synthesis is
increased in the retina of diabetic patients with background
retinopathy (34). Vasoactive factors like endothelins (ETs), by virtue of their extensive tissue distribution and widespread biological actions, are important mediators of pathogenic changes in
several diseases affecting microvasculature, including diabetes. We
have previously demonstrated that ET-1 and ET-3 expressions are
upregulated in the retina of both diabetic and galactose-fed rats
(9, 11). We have further demonstrated that ET
receptor blockade prevents hyperhexosemia-induced
vasoconstriction, increased ECM production, and BM thickening in the
retina and glomeruli of diabetic rats (7, 9, 10, 11). We
have shown that diabetes-induced myocardial focal scarring and
increased ECM protein production may be blocked by ET antagonism
(8). The major ET isoforms having biological significance
with respect to diabetic complications are ET-1 and ET-3 (21,
22). Several pathways involved in diabetes may also result in
increased ET synthesis. Protein kinase C (PKC) activation, secondary to
hyperglycemia, may lead to ET-1 mRNA upregulation (18, 21,
22). We have demonstrated that vascular endothelial growth
factor (VEGF), which is upregulated in diabetes via a PKC-dependent
mechanism, as well as reduced nitric oxide (NO), may lead to ET-1
upregulation in ECs (5). Various pathways of tissue injury
caused by hyperglycemia in vivo or high glucose (HG) concentration in
culture may activate transcription factors such as nuclear factor
(NF)-B and activating protein (AP)-1 (27, 30, 33).
NF-
B and AP-1 regulate expression of several genes important in the
pathogenesis of diabetic complications (27, 30). NF-
B
is present in several cell types, including ECs (38).
Normally, NF-
B exists in an inactive form in the cytoplasm bound to
an inhibitory protein, I
B. Upon stimulation, I
B is hydrolyzed and
the p50/p65 dimer translocates to the nucleus and initiates
transcription (1, 6, 15, 27, 30). Resynthesis of I
B,
induced by NF-
B, allows sequestration of NF-
B in the cytoplasm,
shutting down the NF-
B response (1, 6, 15). ET-1 has
been demonstrated to activate NF-
B in the hepatic stellate cells via
ETB receptor (12). Activation of genes in
different altered physiological and pathological conditions may involve coordinated participation of NF-
B and another transcription
factor, AP-1 (17, 37). AP-1 consists of homodimers of
Jun or heterodimers Fos and Jun
(17, 37). It is also regulated by cellular stress. Furthermore, angiotensin II-induced end organ damage in hypertension has been shown to be mediated via ET receptor-dependent NF-
B and
AP-1 activation (29). Human ECs cultured in the presence of HG (25 mmol/l) upregulated expression of many basement proteins including FN, thus mimicking effects of diabetes (3).
Therefore, in the present study, we sought to determine the role of
NF-
B and AP-1 in mediating HG-induced FN synthesis in ECs in culture and, more importantly, to determine whether such changes are regulated by ETs and their receptors. We used two different types of ECs for our
studies. We used HUVECs (human umbilical vein ECs), because these are
well-characterized ECs, and HMECs (human microvascular ECs), because
diabetic angiopathy involves both microvessels and macrovessels and,
therefore, investigations in both microvascular and macrovascular ECs
are warranted.
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MATERIALS AND METHODS |
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Reagents. All reagents were obtained from Sigma Chemical (St. Louis, MO) unless otherwise mentioned.
Cell culture.
The HUVEC and HMEC lines were obtained from American Type Culture
Collection (Rockville, MD) and Clonetics (Clonetics, Walkersville, Maryland), respectively. These cells were plated at 2,500 cells/cm2 in EC growth medium (EGM) (Clonetics). EGM is a
modified MCDB 131 formulation and is supplied with 10 µg/l human
recombinant epidermal growth factor, 1.0 mg/l hydrocortisone, 50 mg/l
gentamicin, 50 µg/l amphotericin B, 12 mg/l bovine brain extract, and
10% fetal bovine serum. Cells were grown in 25-cm2 tissue
culture flasks. Appropriate concentrations of glucose were added to the
medium when cells were 80% confluent. L-Glucose was used
as a control. In all experiments, the specific ETA blocker TBC11251 (courtesy of Dr. R. Tilton, Texas Biotechnology, Houston, TX)
and the PKA inhibitor N-tosyl-L-phenylalanine
chloromethyl ketone (TPCK) were used at 10 µmol/l (5).
Phorbol 12-myristate 13-acetate (PMA) was used at 60 µg/l
(5). The selective ETB antagonist BQ788, dual
ETA and ETB antagonist bosentan (courtesy of
Dr. M. Clozel, Actelion, Allschwill, Switzerland), and the PKC
inhibitor chelerythrine were used at 1 µmol/l (5).
NF-B inhibitor SN50 and inactive peptide control SN50M (Calbiochem, La Jolla, CA), as well as dual NF-
B and AP-1 inhibitor curcumin, were used at 20 µmol/l (23, 31). ET-1 and ET-3
(Peninsula Laboratories, Belmont, CA) were used at 5 nmol/l, and
NF-
B inhibitor aminopyrrolidine-2,4-dicarboxylate (PDTC) was used at
100 µmol/l (25). All experiments were carried out after
24 h of incubation unless otherwise indicated. Three different
batches of cells, each in duplicate, were investigated.
Cell proliferation and cell viability. Cell viability was measured by trypan blue dye exclusion, and cell proliferation was evaluated by the microculture tetrazolium assay using 2,3-bis(2 methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) (36). Cells were seeded in 100-ml, 96-well plates (3,200 cells/well, 10,000 cells/cm2). After 24 h, experimental agents were applied (100 ml) and the cultures were incubated for 1, 3, or 5 days at 37°C. XTT (50 µg) and 0.38 mg of phenazine methosulfate were added to each well (50 µl) after cell inoculation. The cells were incubated at 37°C for 4 h, and the plates were mixed on a mechanical plate shaker. Absorbance at 450 nm was measured with the Bio-Rad model 3550 microplate reader (Bio-Rad Laboratories, Hercules, CA). All experiments were performed in triplicate.
Preparation of nuclear protein fractions.
Nuclear extracts of HUVECs and HMECs were prepared as described
elsewhere, with some modifications (19, 40). Rapid
detection of octamer binding proteins with "mini-extracts" was
prepared from a small number of cells. Briefly, the cells were washed, resuspended in phosphate-buffered saline, and centrifuged (7,000 g for 15 s). The pellet was resuspended in 0.4 ml of
cold buffer A [10 mmol/l HEPES, pH 7.9, 10 mmol/l KCl, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 1 mmol/l 1,4-dithiothreitol (DTT),and 0.5 mmol/l PMSF] by gentle pipetting. The cells were allowed to swell on ice for 15 min. Twenty-five microliters of a 10% Igepal CA-630 was added, and cells were vortexed vigorously. The homogenate was
centrifuged (10,000 g for 30 s). The nuclear pellet was
resuspended in 50 µl of ice-cold buffer C (20 mmol/l
HEPES, pH 7.9, 0.4 mol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l
DTT, and 1 mmol/l PMSF), and the tube was vigorously rocked at 4°C
for 15 min on a shaking platform. The nuclear extract was centrifuged
at 4°C (15,000 g for 5 min), and the supernatant was
frozen at 70°C. The protein concentrations were measured using the
BCA protein assay, with bovine serum albumin as a standard (Pierce, IL).
Electrophoretic mobility shift assay.
NF-B and AP-1 consensus oligonucleotide (Promega, WI) DNA
probes (Table 1) were prepared by
end-labeling with [
-32P]ATP (Amersham, Quebec, QC,
Canada) using T4 polynucleotide kinase. The probes were purified by
ethanol precipitation and resuspended in 10 mmol/l Tris and 1 mmol/l
EDTA (pH 7.6). Nuclear proteins (5 µg) were incubated with 100,000 cpm of 32P-labeled consensus oligonucleotides for 30 min at
room temperature. The incubation was carried out in a buffer containing
10 mmol/l Tris (pH 7.5), 50 mmol/l NaCl, 1 mmol/l MgCl2, 5% glycerol,
0.05% NP-40, 0.5 mmol/l EDTA, 0.5 mmol/l DTT, and 0.5 µg of
poly(dI-dC). Protein-DNA complexes were resolved on a standard 6%
(NF-
B) and 4% (AP-1) nondenaturing polyacrylamide gel in 0.5× TBE
running buffer. After 0.5 h of electrophoresis at 350 V, gels were
dried under a heated vacuum onto Whatman paper and subjected to
autoradiography (19, 40). Anti-NF-
B (p65) monoclonal
antibody and anti AP-1 (c-jun) polyclonal antibody (Santa
Cruz Biotechnology, CA) were used for supershift assay. The specificity
of binding was further confirmed by incubation with 100-fold unlabeled
oligonucleotides. The blots were quantified by densitometry. The
analyses for comparison of effects of various reagents were carried out
after 4 h of incubation.
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Western blotting. Total proteins were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were analyzed by Western blotting using the antibodies described above. The signals from Western blots were obtained using horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibody (Santa Cruz Biotechnology) and developed using the chemiluminescent substrate (Amersham Pharmacia Biotechnology, Amersham, UK).
RNA isolation. TRIzol reagent (Canadian Life Technologies, Burlington, ON, Canada) was used to isolate RNA. RNA was extracted with chloroform, followed by centrifugation to separate the solution into aqueous and organic phases. RNA was recovered from the aqueous phase by precipitation with isopropyl alcohol and suspended in diethyl pyrocarbonate-treated water.
First strand cDNA synthesis.
First strand cDNA synthesis was performed using Superscript-II system
(Canadian Life Technologies). RNA was added to Oligo (dT) primers
(Canadian Life Technologies), denatured at 65°C, and quenched on ice
for 10 min. Reverse transcription was carried out by the addition of
MMLV-reverse transcriptase and dNTP at 42°C for 50 min in a total
reaction volume of 20 µl. The reaction was terminated by incubating
at 70°C for 15 min. The resulting RT products were stored at
20°C.
Competitive PCR. The amplification of RTs was carried out as previously described with some modifications (5, 7, 9, 10, 11). Competitive PCR was performed for both ET-1 and FN. Competitor DNA fragments were generated using TaKaRa competitor kit. Several dilutions of competitor DNA and the target RT product were mixed to standardize the reaction. The primer sequence and the predicted product size are outlined in Table 1. Reactions were performed in 25-µl volumes containing 1 × PCR buffer, 1.5 mmol/l MgCl2, 250 µmol/l dNTP mix, 1 µmol/l each of amplification primer, 2.5 U Taq polymerase, and 1 µl of RT product. The amplification for FN mRNA (25 cycles) was carried out as follows: 45 s at 94°C (denaturation), 45 s at 54°C (annealing), and 1 min at 72°C (extension). Amplification of ET-1 mRNA was carried out at the same temperatures using 30 cycles. The amplification products were analyzed on a 3% agarose gel in 1 × TBE buffer, stained with ethidium bromide, and visualized under ultraviolet light.
Quantitation. Quantitation was performed by densitometric analysis of the bands using Mocha software (SPSS, Chicago, IL). The densitometric values were expressed as gene to competitor ratio per microgram of total RNA.
Confocal microscopy for NF-B and FN.
Cells were plated on eight chamber-tissue culture slides and incubated
for 24 h. Glucose (25 mmol/l) was added 24 h before different
stimulators and inhibitors were added. After 24 h of stimulation
or inhibition, cells were fixed with 1:1 methanol:acetone. The cells
were then stained using polyclonal rabbit antihuman FN antibody (1/50;
DAKO Diagnostics Canada, Mississauga, ON, Canada) or anti-NF-
B mouse
monoclonal antibody (1/40; Santa Cruz) antibody. Goat
anti-rabbit/anti-mouse IgG labeled with Texas red or FITC (1/100;
Vector Laboratories, Burlington, ON, Canada) was used as the secondary
antibody. Slides were mounted in Vectashield fluorescence mounting
medium with 4',6'-diamino-2-phenylindole (DAPI; Vector Laboratories)
for nuclear staining. Microscopy was performed by an examiner unaware
of the identity of the sample using a Zeiss LSM 410 inverted laser scan
microscope equipped with fluorescein, rhodamine, and DAPI filters (Carl
Zeiss Canada, North York, ON, Canada).
Statistical analysis. Data are expressed as means ± SE and were analyzed by ANOVA followed by Student's t-test with Bonferroni corrections. Differences were considered significant at values of P < 0.05.
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RESULTS |
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Cell proliferation and viability. No difference in viability was noted in cells treated with low (5 mmol/l) or high glucose (25 mmol/l) up to 96 h. The cells in HG, however, showed a significantly (P < 0.001) lower proliferation rate compared with cells in low glucose (data not shown).
ET-1 expression by HG. We then reproduced our previous findings (5) of upregulation of ET-1 mRNA levels in both HUVECs and HMECs exposed to HG in culture medium (data not shown). We also confirmed the inhibitory effects of PKC inhibitor chelerythrine on HG-induced ET-1 gene expression (data not shown).
Modulation of HG-induced FN expression by ET receptor antagonists
and inhibitors of PKC, NF-B and AP-1.
We have previously demonstrated that hyperhexosemia-induced increased
expression of ET is an important mediator of increased ECM production
in retinas, glomeruli, and the hearts of diabetic rats (9, 11,
24, 26, 34). One of the most important ECM proteins that is
overexpressed in these organs in diabetes is FN (3, 9, 35 ). As reported by Cagliero et al. (3), HG
concentrations increase a number of ECM-related genes, including FN. In
the present study, incubation of the HUVECs in HG (25 mmol/l) caused a
time-dependent increase in FN mRNA expression, reaching a maximal
increase after 24 h, and thereafter maintained a similar level at
least up to 72 h (Fig.
1A). Therefore, subsequent
experiments were performed with HG treatment for 24 h in both
HUVECs and HMECs. FN mRNA expression was demonstrated as three bands at
852 base pairs (bp) (EIIIA+), 582 bp (EIIIA
),
and an intermediate band of ~650 bp. The intermediate band represents
a heteroduplex DNA consisting of one strand of each product mentioned
above (35). The competitor band was present at 340 bp. A
similar increase was also observed when both the ECs in 5 mmol/l
glucose were incubated with ET-1 and ET-3 (Fig. 1,
B-D). Failure of mimicry by an equal
concentration of L-glucose suggests a lack of involvement
of hyperosmolality in HG-induced FN overexpression (Fig. 1,
B-D). HG-induced increased FN mRNA accumulation was prevented in both cell types by the nonselective ET
receptor antagonist bosentan, as well as the selective ETA antagonist TBC11251 (Fig. 1, B-D). The
ETB-selective antagonist BQ788 was, however, only effective
in blocking FN mRNA expression in HMECs but not in HUVECs (Fig. 1,
B-D).
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HG concentration or ET can activate NF-B and AP-1 in cultured
ECs.
As previously reported, HG concentration causes NF-
B activation in
bovine aortic ECs (32). In our experiments, incubation of
HUVECs and HMECs in HG caused a time-dependent activation of NF-
B.
In HUVECs, Western blot analysis of total cellular proteins demonstrated maximum increase at 4-8 h. (data not shown). EMSA, however, demonstrated peak NF-
B activation at 24 h of
incubation, which remained at the same level up to 48 h (data not
shown). In HMECs, the peak NF-
B activation was also observed after
24 h of incubation (data not shown). Therefore, subsequent
experiments were performed with 24-h incubation. Similar NF-
B
activation was observed when the cells in 5 mmol/l glucose were
incubated with ET-1, ET-3, or PMA (Figs. 3, A and
B, and 4,
A-C). The specificity of NF-
B activation
was further established by supershift assay (Fig. 3, A and
B) and also in the experiments in which both HG and
ET-induced NF-
B activation were blocked by the NF-
B inhibitor SN50, antioxidant and NF-
B inhibitor PDTC, and dual NF-
B and AP-1
inhibitor curcumin, but not by inactive peptide SN50M (Fig. 4). HG-induced NF-
B activation was
further blocked by the specific ETA antagonist TBC11251 and
by the dual ETA/ETB antagonist bosentan. In
HUVECs, ETA antagonist was more effective than
ETB antagonist, whereas HMECs were equally responsive to
ETA and ETB antagonists (Fig. 4). Similar
inhibition of NF-
B activation was seen when the cells in HG were
incubated with the PKC inhibitor chelerythrine (Fig. 4,
A-C). No effects of PKA inhibitor TPCK were
seen in either of the cells (Fig. 4, A-C).
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DISCUSSION |
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The key findings of the present study are the following:
1) HG-induced accumulation of FN mRNA and protein in human
ECs, requiring activation of NF-B and AP-1, can be prevented by ET
receptor antagonism; 2) these HG-induced effects in human
ECs can be mimicked by activation of ET receptor(s); and 3)
all the observed effects of HG are very similar in both macrovascular
(HUVEC) and microvascular (HMEC) ECs. The only difference in the two
cells we observed is that both ETA and ETB
mediate the ET response in HUVECs, whereas ETA
predominantly mediates the ET response in HMECs. Furthermore, it is
well known that HG activates PKC (16). We studied the importance of the PKC pathway in HG-induced activation of human ECs by
evaluating the countereffect of PKC inhibitor on HG-induced FN
expression and NF-
B and AP-1 activation and by further evaluating the HG-mimicking effects of the PKC activator phorbol ester. Indeed, our results showed the requirement of PKC in HG-induced effects in both
HUVECs and HMECs. Activation of PKC is also required for HG-induced
upregulation of ET in many cells, including ECs (5). Therefore, HG causing upregulation of ET expression through PKC activation can further activate transcription factors NF-
B and AP-1
and thereby increases FN expression. However, hyperhexosemia-induced ET
upregulation can also be controlled by NF-
B and AP-1 (21, 22,
33). Furthermore, as ET receptors are predominantly
Gq-coupled, their activation may lead to activation of
phospholipase C, elevation of [Ca2+]i, and
activation of PKC. Therefore, combining the results of our present
study with previously published studies (5, 21, 22, 33),
we can speculate that PKC, NF-
B, and AP-1 can control both upstream
and downstream effector molecules in the HG-induced signaling pathway.
Gel shift and supershift analyses in this study have shown activation
of at least p65 subunits of NF-B and c-Jun subunits of
AP-1 in nuclear extracts of both HUVECs and HMECs. The specificity of
these bindings has been examined further by competition experiments performed with 100-fold excess unlabeled nucleotides corresponding to
NF-
B and AP-1 binding sequences. Elevated nuclear levels of p65 have
also been observed in HG-treated ECs by Western blot analysis (data not
shown). In hepatoma cells, NF-
B has been shown to play both positive
and negative regulators acting on the FN gene (20).
Although several potential NF-
B binding sites have been identified
in the FN promoter, functional significance of only some of these has
been determined. Regulation of FN gene expression has been shown to be
offered by
41 NF-
B binding site (20). The DNA
sequence between +1 and +136 has been shown to be responsible for part
of PKC-mediated activation of FN gene in hepatoma cells
(20). A potential NF-
B p65 response element present in the FN promoter has been shown to serve as a positive regulator of FN gene expression (20). Identification and
characterization of a NF-
B binding site in the promoter region of FN
gene responsible for ET responsiveness in ECs require further
investigation. An AP-1 binding site mediating angiotensin II-induced
transcriptional activation of FN gene has been shown in its promoter
region (39). Whether ET-induced activation of FN gene also
utilizes the same region of the gene remains to be investigated.
The effects of HG concentration in various cell types may be mediated
by different PKC isoforms. For example, PKC has been shown to
mediate some of the HG-induced effects in porcine aortic ECs
(14). Which PKC isoform(s) mediate(s) the HG-induced
effect on FN expression in HUVECs and HMECs remains to be determined.
Cyclic AMP response element (CRE) present in FN gene promoter is known to play an important role in FN gene expression (2). However, PKA inhibitor did not reverse HG-induced upregulation of FN expression in the present study. Therefore, CRE in FN gene may not play any important role in HG-induced upregulation of FN in HUVECs and HMECs.
In conclusion, using macrovascular (HUVEC) and microvascular (HMEC) EC
lines, we have demonstrated that constitutive expression of functional
ET receptor(s) is required for HG-induced upregulation of FN and that
ETs can mimic this HG-induced effect. Furthermore, both HG- and
ET-induced FN upregulation involve two transcription factors, NF-B
and AP-1. The observations made in the present study are likely to have
relevance to the vasculopathy in diabetes. Studies are underway to
extrapolate our present findings in vivo using models of diabetic complications.
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ACKNOWLEDGEMENTS |
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This study was supported in part by grants from the Canadian Diabetes Association in honor of Margaret Francis and the Canadian Institute of Health Research (MOP 43841).
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Chakrabarti, Dept. of Pathology, Dental Sciences Bldg., Univ. of Western Ontario, London, Ontario, N6A 5C1, Canada (E-mail: schakrab{at}uwo.ca).
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 18, 2002;10.1152/ajpcell.00192.2002
Received 24 April 2002; accepted in final form 16 September 2002.
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