Perinatal Research Centre, Departments of Obstetrics/Gynecology and Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2S2
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ABSTRACT |
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Peroxynitrite, a marker of oxidative
stress, is elevated in conditions associated with vascular endothelial
cell dysfunction, such as atherosclerosis, preeclampsia, and diabetes.
However, the effects of peroxynitrite on endothelial cell function are not clear. The endothelium-derived enzymes nitric oxide synthase (NOS)
and prostaglandin H synthase (PGHS) mediate vascular reactivity and
contain oxidant-sensitive isoforms (iNOS and PGHS-2) that can be
induced by nuclear factor (NF)-B activation. We investigated the
effect(s) of peroxynitrite on NOS and PGHS pathways in endothelial cells. We hypothesized that peroxynitrite will increase levels of iNOS
and PGHS-2 through activation of NF-
B. Western immunoblots of
endothelial cells show that 3-morpholinosydnonimine (SIN-1; 0.5 mM), a
peroxynitrite donor, increased iNOS protein mass, which can be
inhibited by pyrroline dithiocarbamate (an NF-
B inhibitor) (167 ± 24.2 vs. 78 ± 19%, P < 0.05, n = 6). SIN-1 treatment also significantly increased
NF-
B translocation into endothelial cell nuclei (135 ± 10%,
P < 0.05). Endothelial NOS, PGHS-1, and PGHS-2 protein
levels were not altered by SIN-1. However, prostacyclin synthase
protein mass, but not mRNA, was significantly reduced in SIN-1-treated
endothelial cells (78 ± 8.9%, P < 0.05). Our results illustrate novel mechanisms through which peroxynitrite may
modulate vascular endothelial function.
oxidative stress; vascular function
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INTRODUCTION |
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OXIDATIVE STRESS is intimately linked to the progression of many diseases such as preeclampsia (6), diabetes (39), and atherosclerosis (22). These conditions share widespread vascular abnormalities, which is likely the result of endothelial cell dysfunction. The mechanism(s) by which oxidative stress mediates endothelial cell function, and ultimately vascular reactivity, is not fully understood. However, one manifestation of oxidative stress, the formation of peroxynitrite, is hypothesized to be involved in the progression of a variety of diseases (3).
Under conditions of oxidative stress, the oxygen free radical, superoxide anion, will preferentially react with available nitric oxide rather than its endogenous neutralizer superoxide dismutase, thus increasing the formation of peroxynitrite in tissues (3). Peroxynitrite formation has been observed in the maternal vasculature of women with preeclampsia (29), in the placental vasculature of women with preeclampsia and women with diabetes (23), and in platelets from patients with diabetes (38) as well in atherosclerotic plaques (2). The localization of peroxynitrite to these tissues suggests that it may be involved in altering vascular function. However, the direct effect(s) of peroxynitrite on endothelial cells is largely unknown.
The endothelium has an important function in maintaining vascular tone,
which is mediated in part by the enzymes nitric oxide synthase (NOS)
and prostaglandin H synthase (PGHS). NOS catalyzes the reaction of
L-arginine to nitric oxide, whereas PGHS uses arachidonic
acid as a substrate, forming prostaglandin H2
(PGH2). PGH2 is converted to vasoactive
molecules, such as prostacyclin and thromboxane, via specific synthases
(prostacyclin synthase and thromboxane synthase, respectively). Both
NOS and PGHS have inducible isoforms (iNOS and PGHS-2), which are
oxidant sensitive through the activation of nuclear factor-B
(NF-
B) (11, 30).
The involvement of NOS and PGHS in altering vascular function has been implicated in conditions characterized by oxidative stress (3, 36). Enhanced NOS activity in an environment of oxidative stress would result in scavenging of NO by superoxide anion, forming the potent pro-oxidant peroxynitrite, thus reducing nitric oxide bioavailability as a vasodilator (3). PGHS-2 also may mediate vascular dysfunction in conditions characterized by oxidative stress. For example, in carotid arteries and macrophages from patients with atherosclerosis, PGHS-2 expression is elevated (2, 36). Furthermore, the enzymatic activity of prostacyclin synthase is inhibited by the pro-oxidant peroxynitrite, which could result in reduced prostacyclin-mediated vasodilation (44). Thus endothelial cells maintain a balance of vasodilators and vasoconstrictors, in part through NOS- and PGHS-dependent mechanisms, which may be disrupted by oxidative stress. However, the involvement of peroxynitrite in regulating these pathways has not been well elucidated.
The aim of this study was to determine the effect of peroxynitrite on
endothelial cell function, focusing on pathways that modulate vessel
reactivity. We hypothesized that peroxynitrite would increase the
levels of iNOS and PGHS-2, through the activation of NF-B, and
decrease protein levels of prostacyclin synthase. The results from this
study will help determine the mechanisms by which peroxynitrite may
alter endothelial cell function, leading to the vascular abnormalities
that are characteristic of patients with diabetes, atherosclerosis, and preeclampsia.
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MATERIALS AND METHODS |
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Reagents.
-Minimum essential medium (
-MEM) with or without phenol red,
horse serum, L-glutamine, trypsin, phosphate-buffered
saline (PBS), and penicillin/streptomycin were purchased from GIBCO
(Gaithersburg, MD). Nystatin, 3-morpholinosydnonimine
N-ethylcarbamide (SIN-1), pyrroline dithiocarbamate (PDTC),
gentamycin, kanamycin, and bovine serum albumin (BSA) were purchased
from Sigma (Oakville, ON, Canada).
Endothelial cell culture.
Bovine microvascular endothelial cells were selected for our study
because they originate from a resistance-sized vascular bed and
therefore intrinsically function to regulate systemic vascular
resistance. When cultured, these cells show typical endothelial cell
characteristics: growth in a monolayer, cobblestone morphology, and
positive detection of von Willebrand factor. Cells were grown at 37°C
in an atmosphere of 5% CO2-95% air. Growth media
(-MEM) were supplemented with 1% L-glutamine, 10%
horse serum, 5 µg/ml gentamycin, 20 µg/ml kanamycin,
penicillin-streptomycin, and nystatin. At confluence, cells were plated
into six-well plates (106 cells/well) in a volume of 1 ml.
After 24 h, cells were quiesced with phenol red-free media
overnight. Before stimulation, plates were replaced with fresh media.
After experimentation, the total protein content of the cells was
determined using the Bradford Method (4) with BSA used as
a standard.
Experimental protocol.
Our experiments involved the use of SIN-1, a peroxynitrite donor that
breaks down at physiological pH to form nitric oxide and superoxide
anion simultaneously (12). SIN-1 is commonly used as an
effective pharmacological agent for administering peroxynitrite at the
level of the cell in a relatively stable form, as opposed to using
authentic peroxynitrite, which is highly volatile and may decompose
into inactive metabolites before reaching the cell. We determined the
formation of peroxynitrite in our cells by detection of nitrotyrosine,
using both Western immunoblots and immunocytochemistry (Fig.
1). Furthermore, to confirm that the
effects of SIN-1 were mediated by peroxynitrite, we studied the effect
of exogenous peroxynitrite (Cayman Chemicals, Ann Arbor, MI) as well as
the effect of the nitric oxide donor sodium nitroprusside (Sigma) on
endothelial cell function.
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Nuclear protein preparation.
At confluence, cells were quiesced for 24 h in a T-75 flask.
Afterward, cells were stimulated with 0.5 mM SIN-1 for 4 h. This time frame was chosen on the basis of previous accounts of NF-B activation (8). Nuclear protein was extracted from the
cells by following the method described by Schreiber et al.
(31). Briefly, cells were washed with 10 ml of
Tris-buffered saline (TBS) and pelleted by centrifugation at 1,500 g for 5 min. The pellet was resuspended in 1 ml of TBS and
pelleted again by spinning in a microfuge for 12,000 g for
15 s. The pellet was then resuspended in cold buffer A
[10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM
dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride
(PMSF)]. The cells were allowed to swell on ice for 15 min, and then
25 µl of 10% Nonidet P-40 (Boehringer Mannheim, Mannheim, Germany)
were added. The tube was vortexed for 10 s and centrifuged for
30 s at 12,000 g. The nuclear pellet was resuspended in
50 µl of ice-cold buffer B (20 mM HEPES, 0.4 M NaCl, 1.0 mM EDTA, 1.0 EGTA, 1.0 mM DTT, and 1.0 mM PMSF). The tube was agitated for 15 min at 4°C and then centrifuged for 5 min, and the supernatant containing the nuclear protein was stored at
80°C for future use.
Western immunoblotting.
Western immunoblots were performed to measure levels of endothelial NOS
(eNOS), inducible NOS (iNOS), PGHS-1, PGHS-2, prostacyclin synthase,
and nitrotyrosine in endothelial cells and NF-B in endothelial cell
nuclei. Samples were diluted 1:4 with sample buffer (1.0 M Tris-Cl,
glycine, 2% SDS, 2% bromphenol blue, and
-mercaptoethanol) and
boiled for 3 min. Protein from each sample was loaded (8 µg/well),
and 10 µl of Kaleidoscope molecular weight marker (Bio-Rad, Hercules,
CA) were loaded in a separate well to allow for accurate determination
of molecular weight. eNOS and iNOS proteins were run on an 8%
acrylamide gel, whereas PGHS-1, PGHS-2 , prostacyclin synthase,
NF-
B, and nitrotyrosine were run on a 10% gel. Protein was
separated by electrophoresis at 120 V for 1.5 h in a mini-gel
apparatus after the procedure of Laemmli (19).
Immunocytochemistry.
Cells were plated onto 22 × 22-mm glass coverslips and treated
with 1.0 mM SIN-1 for 6 h. Afterward, the coverslips were fixed with 10% formalin-phosphate and stored overnight at 4°C.
SIN-1-treated cells were incubated with 1:100 rabbit polyclonal
anti-NF-B (Santa Cruz) or 1:100 monoclonal anti-nitrotyrosine
(Transduction Laboratories) overnight at 4°C. A standard
immunostaining protocol was followed by using the Vectastain ABC kit
from Vector Laboratories (Burlingame, CA).
Total RNA isolation. After stimulation, cells were lysed directly in a six-well plate by adding 1 ml of Trizol reagent (GIBCO, Burlington, ON, Canada), passing the cell lysate through a pipette several times, and allowing the cells to sit for 5 min at room temperature. The lysate was transferred into Eppendorf tubes, 0.2 ml of chloroform was added, and each tube was vigorously shaken. Tubes were centrifuged for 3 min at 12,000 g at 4°C. The aqueous phase was transferred into a fresh tube, 0.5 ml of isopropanol was added, and tubes were incubated at room temperature for 10 min. Afterward, the tubes were centrifuged at 4°C at 12,000 g for 10 min. The RNA pellet was washed with 75% ethanol and resuspended in 50 µl of TE buffer (10 mM Tris-Cl, pH 7.5, and 1.0 mM EDTA, pH 8.0). The RNA concentration was determined by measuring the absorbency at 260 nm and calculating the optical density.
Real-time PCR. First-strand cDNAs were synthesized by incubating 1.0 µg of total RNA from endothelial cells with 1.0 µM random primers (Stratagene, La Jolla, CA), in a 20-µl reaction volume containing cDNA buffer (50 mM Tris-Cl, 75 mM KCl, 3 mM MgCl2, and 5 mM DTT), 2.5 mM deoxynucleotide triphosphates (dNTPs), and 1 unit of reverse transcriptase (Superscript II; GIBCO). The mixtures were incubated for 50 min at 48°C, followed by 5 min at 85°C. The PCR reaction contained 25 µl of the SYBR green Master Mix Kit (containing DNA polymerase, dNTPs, and MgCl2; Applied Biosystems) 100 nM sense and antisense primers, and 2 µl of the reverse transcriptase reaction. The temperature profile was 3 min at 95°C followed by 38 cycles of 30 s at 95°C and 30 s at 60°C (or 30 s at 53°C for cyclophilin, a housekeeping gene). The following primers were used for bovine prostacyclin synthase: forward, 5'-AGGATGAAGGAGAAGCATGG-3', and reverse, 5'-GGGCTCCTCGAGTTCTCCTA-3'. The following primers were used for cyclophilin: forward, 5'-CACCGTGTTCTTCGACATCAC-3', and reverse, 5'-CCA- GTGCTCAGAGCTCGAAAG-3'.
Data analysis.
Western immunoblot density was quantified using the Fluor-S Max
Quantity One software (Bio-Rad). Protein values are percent control
expressed as means ± SE. RNA content was measured and quantified
using the I-Cycler software (Bio-Rad). The starting quantity of RNA was
determined on the basis of the number of cycles required to amplify the
cDNA above a set threshold. Starting quantity values (for both
cyclophilin and prostacyclin synthase) were obtained from a standard
curve, created from control RNA (ranging from 1.0 to 1,000 ng RNA), and
then normalized to cyclophilin RNA for each experimental group. Data
are standardized amount of RNA per amount of cyclophilin RNA, expressed
as means ± SE. Either Student's t-test (for
comparison between 2 groups) or one-way ANOVA followed by a Fisher
least significant difference post hoc test was used to determine
statistical significance between groups (P < 0.05). Immunocytochemistry of NF-B and nitrotyrosine immunostaining was
qualitatively described.
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RESULTS |
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Effect of a peroxynitrite donor on iNOS protein levels and NF-B
activation in endothelial cells.
We investigated the effect of peroxynitrite on endothelial cell enzymes
that are important for vascular function and known to be oxidant
sensitive. In this study SIN-1 was used as an endogenous peroxynitrite
donor (24, 25, 42). Cell damage was assessed using a LDH
assay and was negligible at all doses.
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Effect of a peroxynitrite donor on PGHS-2 and prostacyclin synthase
expression.
We also studied the effect of peroxynitrite on the enzymes PGHS-1,
PGHS-2, and prostacyclin synthase. Contrary to our hypothesis, treating
endothelial cells with a peroxynitrite donor did not increase PGHS-2
protein levels (104 ± 8.7%, P < 0.05; Fig.
5A). The constitutively
expressed PGHS-1 also was not altered by SIN-1 treatment (Fig.
5B). On the other hand, prostacyclin synthase, the enzyme
downstream of PGHS that forms the vasodilator prostacyclin, was
significantly inhibited by SIN-1 treatment of the endothelial cells
(78 ± 8.9%, P < 0.05; Fig.
6).
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Effect of authentic peroxynitrite on iNOS and prostacyclin
synthase.
To confirm the cellular effects of peroxynitrite formed by SIN-1, we
treated endothelial cells with 100 µM authentic peroxynitrite for
6 h. Our data show that the same changes in protein levels occur
with peroxynitrite treatment as with SIN-1. For example, iNOS protein
levels increased (133 ± 9%, P = 0.07; Fig.
8A), whereas prostacyclin
synthase was significantly reduced (73 ± 5.5%, P < 0.05, Fig. 8B). Therefore, these observations support the
results obtained with the peroxynitrite donor SIN-1 and strongly
suggest that peroxynitrite is capable of mediating changes in protein levels in the endothelium.
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DISCUSSION |
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This study shows that peroxynitrite alters endothelial cell
pathways, which are important for vascular function. We have shown, for
the first time, that the peroxynitrite donor SIN-1 significantly upregulates iNOS protein mass in endothelial cells. This novel finding
has important implications for the development of vascular dysfunction,
especially in diseases characterized by oxidative stress. By increasing
iNOS levels in the vasculature, peroxynitrite is capable of stimulating
large quantities of nitric oxide, which, in an environment of oxidative
stress, will rapidly be scavenged, forming more peroxynitrite and
culminating in a vicious positive feedback cycle (Fig.
9). Thus the detrimental effect(s) of
peroxynitrite may be exacerbated by this feed-forward mechanism, in
addition to reducing the bioavailability of nitric oxide as a potent
vasodilator.
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A role for iNOS in vascular pathophysiology is supported by data in the spontaneously hypertensive rat, showing a reduction in the development in hypertension by a specific iNOS inhibitor (13). Furthermore, patients with atherosclerosis show elevated iNOS in coronary plaques, which colocalizes with nitrotyrosine, a marker for peroxynitrite (7). Our data using endothelial cells suggest that peroxynitrite induces iNOS protein. However, it also has been reported that peroxynitrite inhibits iNOS activity in lung epithelial cells (27). This is one potential mechanism by which peroxynitrite may counterbalance the positive feedback cycle, as discussed above. Moreover, the increased expression of iNOS by peroxynitrite also may help explain paradoxical observations in women with preeclampsia; while nitric oxide-mediated vasorelaxation is reduced in maternal blood vessels (1), there is reportedly either no change (5) or elevated (32) levels of nitric oxide metabolites. Thus the impaired endothelium-dependent relaxation, with no concomitant decrease in nitric oxide per se, suggests that there is increased nitric oxide scavenging by oxygen free radicals. Indeed, we have reported evidence of peroxynitrite formation in the vasculature of women with preeclampsia (29).
Currently, little is known about the effect(s) of peroxynitrite on
cellular signaling pathways; therefore, we studied the mechanism by
which peroxynitrite is altering the expression of iNOS. Because NF-B
is an oxidant-sensitive transcription factor, we investigated whether
the pro-oxidant peroxynitrite could potentially activate NF-
B. Using
Western blot analysis of cell nuclei, a technique used by others
(34), we showed that SIN-1 treatment was capable of
increasing NF-
B levels in the endothelial cell nuclei. These data
provide strong correlative evidence that peroxynitrite can induce
nuclear translocation of NF-
B. Our results are further supported by
immunocytochemical data, illustrating diffuse cytosolic immunostaining
in control cells but intense nuclear staining in SIN-1-treated cells.
Furthermore, the peroxynitrite-induced increase in iNOS protein levels
was diminished in the presence of a pharmacological NF-
B inhibitor,
PDTC. Together, our data support the hypothesis that peroxynitrite is a
potential activator of NF-
B in endothelial cells.
The activation of NF-B by peroxynitrite has been implicated in other
cell types. In lipopolysaccharide-stimulated human whole blood,
peroxynitrite can induce interleukin-8 gene expression, which is
blocked by the NF-
B inhibitor PDTC (9). Additionally, during hepatocyte isolation, NF-
B activation can be inhibited by
both NOS blockade (nitro-L-arginine methyl ester) or
through administration of the antioxidant/peroxynitrite scavenger
Trolox (28). In rat lung epithelial cells, SIN-1 activated
a NF-
B-dependent luciferase reporter vector after 8 h of
stimulation (14). Therefore, the pro-oxidant peroxynitrite
is likely able to activate NF-
B, and our data extend this mechanism
to include endothelial cells.
Peroxynitrite previously has been shown to activate PGHS
(10). In our study, we investigated whether peroxynitrite
could increase protein expression. We were unable to detect a
statistically significant increase in PGHS-2 levels in endothelial
cells treated with SIN-1. Because our data suggest that peroxynitrite
activated NF-B in the endothelial cells, it is possible that other
nuclear factors are necessary for full activation of PGHS-2 expression, including high-mobility group protein I (Y), which is required for
complete upregulation of PGHS-2 under hypoxic conditions
(15). Therefore, the formation of peroxynitrite in vivo
may act in concert with other physiological events that were not
present in our experiments to increase PGHS-2 production in endothelial cells.
Prostacyclin synthase, an enzyme downstream of PGHS, was significantly reduced in endothelial cells treated with SIN-1. However, this effect of peroxynitrite on prostacyclin synthase does not seem to be at the level of transcription, because we did not observe any effect of SIN-1 on prostacyclin synthase mRNA expression. One possible mechanism by which peroxynitrite may decrease prostacyclin synthase protein mass is through increasing proteolytic degradation of the enzyme. Zou et al. (45) have illustrated that peroxynitrite inhibits the activity of prostacyclin synthase through a tyrosine nitration-dependent mechanism. Furthermore, recent findings show that nitration of tyrosine residues in certain proteins can increase proteolytic degradation by enhanced targeting to the proteosome (33). Thus prostacyclin synthase is a potential candidate for enhanced proteolytic degradation by peroxynitrite-induced tyrosine nitration.
The effects of peroxynitrite on the PGHS pathway illustrate another important mechanism by which peroxynitrite can alter endothelial cell function. We have shown, for the first time, that peroxynitrite is capable of inhibiting prostacyclin synthase at the level of protein, in addition to the well-documented effects of peroxynitrite on prostacyclin synthase activity (Fig. 9) (45). Furthermore, peroxynitrite has been shown to increase PGHS activity (10). Therefore, in endothelial cells, peroxynitrite formation shifts the balance away from the vasodilator prostacyclin and toward the vasoconstrictors PGH2 and thromboxane. Again, this theory correlates well with data from women with preeclampsia, who show reduced levels of prostacyclin and elevated levels of thromboxane metabolites (41). Furthermore, in atherosclerosis, PGHS-2 is highly expressed and colocalizes with iNOS and nitrotyrosine (2). Finally, PGHS-2-mediated vasoconstriction is more pronounced in aging (37), which also is a state of oxidative stress in which peroxynitrite levels have been shown to be elevated (40).
Although it has been proposed that peroxynitrite may have physiological
effects similar to those of nitric oxide in certain biological systems
(20), our results are likely specific to peroxynitrite.
There are reports illustrating nitric oxide-dependent modulation of
NF-B; however, most of these show an inhibitory effect of nitric
oxide on NF-
B activity (16, 35). Furthermore, for
comparative purposes, we observed that a nitric oxide donor did not
increase iNOS in our endothelial cells (data not shown), whereas
authentic peroxynitrite induced changes in protein levels similar to
those of SIN-1. Therefore, in an endothelial cell culture model, we
find that SIN-1 is an effective, endogenous peroxynitrite donor that
can alter intracellular enzyme expression.
In conclusion, it is well documented that peroxynitrite is elevated in
the vasculature of many conditions characterized by oxidative stress.
Peroxynitrite is increased in the maternal vasculature of women with
preeclampsia (29) and in patients with diabetes as well as
patients with atherosclerosis (2, 38). In addition, in the
placental blood vessels of women with diabetes and women with
preeclampsia, peroxynitrite levels are increased (23), which correlates with the vascular dysfunction and reduced placental perfusion (17). Furthermore, in
ischemia-reperfusion injury, peroxynitrite mediates coronary
vasospasm in bovine coronary arteries (43). Although
peroxynitrite is hypothesized to be involved in the vascular
pathophysiology of these conditions, few studies have focused on the
effects of peroxynitrite on endothelial cell pathways that regulate
vessel function. This study shows that peroxynitrite is a novel
mediator of endothelial cell function. By activating NF-B, thus
increasing the expression of iNOS and inhibiting prostacyclin synthase,
peroxynitrite can contribute to the altered vascular reactivity in a
variety of conditions in which the clinical manifestations are mediated
by oxidative stress.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Canadian Institute of Health Research and the Heart and Stroke Foundation of Alberta. C. M. Cooke is supported by a graduate studentship from the Canadian Institute of Health Research and Alberta Heritage Foundation for Medical Research. S. T. Davidge is an Alberta Heritage Foundation for Medical Research and a Heart and Stroke Foundation of Canada scholar.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. T. Davidge, Perinatal Research Centre, 232 HMRC, Depts. of Obstetrics/Gynecology and Physiology, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2S2 (E-mail: sandra.davidge{at}ualberta.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 October 10, 2001; 10.1152/ajpcell.00295.2001
Received 28 June 2001; accepted in final form 17 October 2001.
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