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Perinatal Research Laboratories, Departments of Obstetrics/Gynecology (I.M.B., J.A.S., T.D., J.M.C., J.Z., R.R.M.) and Meat/Animal Science (R.R.M.), University of Wisconsin, Madison, Wisconsin 53715; and the Center for Perinatal Biology, Loma Linda University School of Medicine (L.Z.), Loma Linda, California 92350
Address all correspondence and requests for reprints to: Ian M Bird, Ph.D., Department Obstetrics and Gynecology, Perinatal Research Laboratories, University of Wisconsin, 7E Meriter Hospital/Park, 202 South Park Street, Madison, Wisconsin 53715. E-mail: imbird{at}facstaff.wisc.edu
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Introduction |
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In initial characterizations of both cPLA2 and eNOS, the hormone-sensitive steps in the production of PGI2 and NO, respectively, it was discovered that both enzymes require Ca2+ for activity. Phosphorylation of cPLA2, however, can also result in a dramatic increase in Ca2+ sensitivity and thus a parallel shift of the Ca2+ dose response to the left, resulting in enhanced cPLA2 activation. Therefore, phosphorylation alone can result in a marked increase in activity without additional increases in free Ca2+. Studies performed in bovine aorta endothelial cells have shown clearly that this occurs and that cPLA2 is indeed a substrate for ERK-1/2, prototypical members of the mitogen-activated protein kinase family (5). In addition, site-directed mutagenesis studies have suggested that phosphorylation of amino acid Ser505 is critical because its elimination negates this response in transfected Chinese hamster ovary (CHO) cells (6). It is therefore apparent that cPLA2 can provide a point of convergence for control of PGI2 production through agonists that act through mobilization of Ca2+ with those that activate ERK-1/2.
The situation with eNOS is more complicated than that for cPLA2 and clearly less well understood. It is apparent that eNOS can be activated by Ca2+/calmodulin and that subsequent total removal of Ca2+ from the medium abolishes such activation of eNOS in vitro. It has also become increasingly clear, however, that either inhibition of tyrosine phosphatases (7) or shear stress (8) can increase eNOS phosphorylation and activity independently of an increase in Ca2+ in endothelial cells (7, 8). The eNOS protein sequence contains a number of putative phosphorylation sites, and although it is not known whether ERK-1/2 or indeed other members of the mitogen-activated protein kinase family directly regulate this enzyme activity, it seems likely that eNOS is another point of convergence of Ca2+ and protein kinase signaling. It also follows that both activation of PGI2 production and NO production could be elicited through agonist-induced elevation of Ca2+ or by activation of endogenous protein kinases (probably ERK-1/2) in UA endothelial cells (UAEC). Until now no in vitro model has been available to investigate the molecular mechanisms underlying the pregnancy-induced changes in vasodilator production in UA endothelium. In this study we describe for the first time a newly developed UAEC culture model derived from nonpregnant (NP) and pregnant (P) ewes that retains the functional differences observed in vivo and further investigated whether the previously reported pregnancy-induced increase in UA vasodilator production from UAEC in response to a variety of agonists is associated with an alteration in receptor coupling to ERK-1/2 and/or intracellular Ca2+. Our results suggest that changes in cell signaling do indeed occur in response to pregnancy and that differential control of vasodilator production relates to differential activation of ERK-1/2 as well as other intracellular kinases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Freshly isolated cells (passage 0) were plated to 35-mm dishes
in MEM containing 20% FBS, 1% penicillin-streptomycin, and 1%
geneticin (growth medium; used throughout). Cells were grown for 6 days
and passaged (passage 1) to 60-mm dishes. Cells were grown to
approximately 70% confluence and then passaged (passage 2) to T75
flasks. Cells were again grown to approximately 70% confluence and
passaged once more (passage 3) to medium containing 10%
dimethylsulfoxide and frozen in liquid nitrogen for long term storage.
Cells were later recovered and grown in T75 flasks to about 70%
confluence and subcultured for experimental use or analyzed for protein
or messenger RNA (mRNA) content (passage 4).
Western analysis
Freshly isolated and cultured UAEC were solubilized directly
into lysis buffer [150 mM NaCl, 50 mM
Tris-HCl, 10 mM EDTA (pH 7.4), 0.1% Tween-20, 0.1%
ß-mercaptoethanol, 0.1 mM pheylmethylsulfonylfluoride, 5
µg/ml leupeptin, and 5 µg/ml aprotinin]. Solubilized protein was
quantified using a modified Lowry assay procedure (Bio-Rad Laboratories, Inc., Hercules, CA). Proteins (10 µg/lane) were
then separated by size on 7.5% polyacrylamide gels (100 V, 2.5 h;
Mini Protean II, Bio-Rad Laboratories, Inc.) alongside
positive controls (adrenal cortex homogenate, 10 µg/lane) and Rainbow
molecular weight markers (Bio-Rad Laboratories, Inc.)
before transfer to Immobilon P membranes (100 V, 2 h). The
Immobilon P membranes were then probed using the enhanced
chemiluminescence reagent detection system, as described by
Amersham Pharmacia Biotech (Arlington Heights, IL), and
exposed to Hyperfilm (Amersham Pharmacia Biotech).
Antiserum incubations were all performed at room temperature diluted as
follows: AT1-R (sc-306, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), diluted 1:750, 2 h followed by
donkey antirabbit horseradish peroxidase (HRP)-linked
Fab2 (Amersham Pharmacia Biotech) at
1:2,500 dilution, 1 h; eNOS (N30020, Transduction Laboratories, Inc., Lexington, KY), diluted 1:750, 2 h,
followed by Amersham Pharmacia Biotech sheep antimouse Ig
HRP (Amersham Pharmacia Biotech), diluted 1:3,000, 1
h; cPLA2 sc-454 (Santa Cruz Biotechnology, Inc.), at 1:100 dilution, 2 h, followed by sheep antimouse
HRP-linked Fab2 (Amersham Pharmacia Biotech), diluted 1:2,500, 1 h; COX-1 (monoclonal, Cayman),
diluted 1:3,000, 2 h, followed by sheep antimouse HRP-linked
Fab2 (Amersham Pharmacia Biotech),
diluted 1:5,000, 1 h; and PGI2 synthase
(PGIS; polyclonal, Cayman) at 1:30,000 dilution, 2 h, followed by
donkey antirabbit HRP-linked Ig (Amersham Pharmacia Biotech), diluted 1:2500, 1 h. Results in each case were
quantified by scanning densitometry (670 scanning densitometer,
Bio-Rad Laboratories, Inc.) and expressed relative to
standards on the same blot. All results were within the linear range
for the antiserum and film in each case.
Extraction of total cellular RNA
Freshly isolated cells or cultured UAEC were washed in fresh
medium 199, and total RNA was extracted using a guanidinium
isothiocyanate/phenol/chloroform method. Cells were solubilized
in 1 ml RNAzol B (Cinna Biotecx Laboratories, Inc.,
Houston, TX). After the addition of 150 µl chloroform and phase
separation by centrifugation (12,000 x g, 20 min), the
upper aqueous phase was removed, extracted twice with
phenol/chloroform/isoamyl alcohol using heavy grade phase lock gel
(5-Prime, 3-Prime, Boulder, CO), and finally mixed with 110% (vol/vol)
isopropanol. RNA was then precipitated by standing at -20 C for 1
h before recovery by centrifugation (12,000 x g, 30
min) and washing of the pellet in 75% ethanol. RNA was
solubilized in molecular biology grade water (5-Prime, 3-Prime) and
quantified by spectrophotometry.
RT/PCR assay for AT11-R mRNA, eNOS mRNA,
and COX-1 mRNA
AT1-R mRNA, eNOS mRNA, and COX-1 mRNA
levels were separately quantified by coupled RT-PCR amplification in
single tube assays using AMV reverse transcriptase and Taq
polymerase and normalized to the 28S ribosomal RNA level, exactly as we
previously described (1, 3, 9, 10). Total cellular RNA (1 µg/tube for
AT1-R or 0.1 µg for eNOS or COX-1 assay) was
incubated in a 50-µl final volume of the RT-PCR reagents. Targets
were designed such that the final products, by homology to the human
and bovine sequences, spanned at least two intron sites in each case;
thus, genomic contamination would not result in a false signal because
it would be of considerably greater size. A standard curve containing
known copy numbers of complementary DNA target sequence was run in each
assay [note that the presence or absence of mRNA species other than
the reverse primer target has little effect on the standard curve and
so was not necessary for the standards (Wiltbank, M., personal
communication)]. At the end of the assay 10 µl of products were
separated by size on a Tris-acetate EDTA/agarose gel and
transferred to MagnaGraph hybridization membrane (Molecular
Separations, Inc., through Fisher Scientific, Westboro,
MA) for Southern blotting against a probe encoding the same
coding sequence, generated by asymmetric PCR (11, 12). After
hybridization, membranes were washed once in 2 x SSC (standard
saline citrate)-0.1% SDS for 30 min and twice in 0.1 x SSC-0.1%
SDS (30 min each time) before drying and direct exposure to a
phosphorimager (Bio-Rad Laboratories, Inc., BI screen; 15
min to 1 h) for direct quantification (Molecular Analyst version
1.4, Bio-Rad Laboratories, Inc.). Data were calculated
from the standard curve as copy number of mRNA per µg total cellular
RNA. All data were normalized to the 28S ribosomal RNA content of each
sample, determined by slot blot analysis of 1 µg total RNA (10).
Acetylated low density lipoprotein (LDL) uptake
UAEC were subcultured onto eight-well glass slides and incubated
for 4 h in the presence of acetylated LDL (10 µg/ml) in growth
medium or in growth medium alone (control). Cells were then thoroughly
washed in serum-free medium and viewed on a Carl Zeiss
axioscope (New York, NY) using a rhodamine excitation/emission filter
set.
Immunocytochemistry
Subconfluent UAEC cultured in eight-chamber slides (Nunc, Inc.,
Naperville, IL) were fixed in 4% formaldehyde after being rinsed in
PBS [10 mM phosphate and 0.14 M NaCl (pH 7.3) containing
0.3% Triton X-100]. Immunolocalization was accomplished using rabbit
antirat-AT1-R and antihuman-Flt-1 antisera
(polyclonal, Santa Cruz Biotechnology, Inc.) as well as
mouse anti-FGF receptors 1 and 3 (monoclonal, Zymed Laboratories, Inc., San Francisco, CA) and epidermal growth factor (EGF)
receptor (EGFR; monoclonal, NeoMarkers, Fremont, CA) antisera.
Localization of specific staining was visualized by indirect
immunoperoxidase detection via the avidin-biotinylated peroxidase
complex method with 3,3'-diaminobenzidine as the chromogen, as
previously described (1, 2, 3, 13). Controls consisted of replacing the
primary antibody with preimmune rabbit and mouse IgG (Vector Laboratories, Inc., Burlingame, CA) at the same concentration as
the primary antibody. After immunostaining, the cells were
counterstained briefly (30 sec) with Harris’ hematoxylin to visualize
nuclei.
Cell mitogenesis assay
Briefly, UAEC were precultured (37 C, 95% air-5%
CO2) overnight in 96-well plates (5,000
cells/well) in 0.2 ml DMEM containing 10% FBS, 10% calf serum, and
1% penicillin-streptomycin. After preculture, media were changed to
0.2 ml serum-free DMEM alone (control) or containing bovine bFGF, human
recombinant EGF, and human recombinant vascular endothelial growth
factor (VEGF; amino acids: 165; R\|[amp ]\|D Systems,
Minneapolis, MN) at 0.01, 0.1, 1, 10, or 100 ng/ml (6
wells/concentration for each growth factor). Controls were run on each
plate. After an additional 72 h of culture, the number of cells
per well was determined by quantification of histone protein as
previously described (14). Wells containing known cell numbers (0,
5,000, 10,000, 20,000, or 40,000 cells/well; 6-well/cell density) were
treated in a similar fashion to establish standard curves.
Agonist-stimulated production of NO and
PGI2
Cells plated in 12-well dishes were washed twice with Krebs
buffer before incubation for 1 h in 450 µl Krebs buffer/well.
Agonists were then added as a 50-µl volume in wells, and incubation
was continued for 1 h. Medium was then collected for assay, wells
were drained thoroughly, and cells were solubilized in lysis buffer for
protein assay. NO production/release were measured immediately by
conversion of total nitrate and nitrite back to NO and electrochemical
detection using a Seivers model 280 NO analyzer (100 µl medium
injected). Results were calculated against a standard curve (using
known quantities of NaNO3). Samples were then
stored frozen for further assay. 6-Keto-PGF1
levels in medium were assayed by enzyme immunoassay using a
commercially available kit (Cayman Chemical Co., Ann Arbor, MI),
using 10 µl medium/well and including quality control standards of
Krebs buffer alone or standards made up in Krebs buffer to monitor
assay drift. Standard curves typically show correlations of
r2 = 0.98.
Fura-2 Ca2+ imaging studies
UAEC were plated to low density (10–20% confluence) on 35-mm
dishes with glass coverslip windows (Intracellular Imaging, Inc., Cincinnati, OH) the night before use to allow attachment.
The next day, immediately before use, cells were loaded with fura-2/AM
for 40 min and rinsed three times in prewarmed (37 C) Krebs buffer
(with 2 mM CaCl2) before covering
them in Krebs buffer (2 ml final volume). Fura-2 loading was verified
by viewing at 380 nm UV excitation on a Nikon Diaphot
inverted microscope (InCyt Im2, Intracellular Imaging, Inc., Cincinnati, OH). A single isolated cell was then set in
the field of view, and recordings commenced using alternate excitation
at 340 and 380 nm at 25-msec intervals and measuring emitted light
using a photomultiplier. From the ratio of emission at 510 nm detected
at the two excitation wavelengths and by comparison to a standard curve
established for the same settings using buffers of known free
Ca2+, the intracellular free
Ca2+ was then calculated in real time using InCyt
Im2 software on-line.
ERK-1/2 phosphorylation assays
UAEC were passaged to 60-mm dishes and maintained for 24 h
before serum withdrawal/incubation in 3 ml MEM for 4 h. Cells were
stimulated with agonists (300 µM ATP, 100 nM
AII, or 10 ng/ml bFGF, VEGF, and EGF) for 10 min, the point at which
ERK-2 phosphorylation was determined to be maximal in preliminary
time-course studies. Reactions were terminated by the addition of
ice-cold PBS. Cells were subsequently washed twice in ice-cold PBS and
solubilized in lysis buffer (4 mM sodium pyrophosphate, 50
mM HEPES (pH 7.5), 100 mM NaCl, 10
mM EDTA, 10 mM sodium fluoride, 2
mM sodium orthovanadate, 1 mM
pheylmethylsulfonylfluoride, 1% Triton X-100, 5 µg/ml leupeptin, and
5 µg/ml aprotinin) before sonication, protein determination
(bicinchoninic acid assay- Sigma, St. Louis, MO), and
Western blotting (10 µg per lane) on 7.5% polyacrylamide gels as
described. Blots were initially probed for total ERK-1/2 protein
(New England Biolabs, Inc., Beverley, MA), which ensures
constant protein expression and also serves as a loading control.
ERK1/2 activity was assessed using a phospho-specific antiserum
(Promega Corp., Madison, WI), which preferentially
recognizes the dually phosphorylated state (pTEpY) of ERK1/2 (dual
phosphorylation of ERK1/2 has been shown to increase the activity of
these enzymes by approximately 1000-fold over that of the basal or
monophosphorylated forms) (15). The intensity of staining was expressed
relative to control.
Statistical analysis
Data were analyzed by one-way ANOVA or Student’s t
test, as appropriate. Data presented are the mean ± 1
SE. Results were considered significant at the
P < 0.05 level.
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Results |
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. Consistent with our previously
reported data (1, 2, 3, 4, 16), we found similar pregnancy-related increases
in the expression of each protein (Fig. 1), demonstrating the
consistency of data using the collagenase dispersion technique compared
with the previously described mechanical endothelial isolation
technique (1, 2).
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). These cells retained
expression of all key proteins at passage 4. Levels of eNOS protein and
mRNA and of COX-1 mRNA still showed higher expression in cells from
pregnant ewes than in cells from nonpregnant ewes at the fourth passage
(Fig. 2), suggesting some retention of the characteristics present at
the time of isolation. The fact that COX-1 protein was not higher in
P-UAEC than in NP-UAEC is probably a reflection of this lower level of
COX-1 protein at passage 4 being near the sensitivity limit for
enhanced chemiluminescence detection, which is to be expected for a
protein with such a short half-life. Both cPLA2
and PGIS expression were also retained at passage 4, but the difference
in between NP- and P-UAEC levels was lost at this time. AT1-R protein
and mRNA levels increased at passage 4 in both NP-UAEC and P-UAEC and
were higher in NP-UAEC at passage 4 than in P-UAEC. This increase is
not unique to UAEC alone, however, in that it has also been reported to
occur in fibroblasts in culture (17).
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), expression of bFGF, EGF, and VEGF
receptors by immunocytochemistry (Fig. 3B), and mitogenic responses to
bFGF, EGF, and VEGF (Fig. 4) were all
retained and similar in NP- and P-UAEC at the fourth passage, as
expected of endothelial cells. In addition, and most important of all,
the functionality of the cells was retained, in that both AII and ATP
enhanced production of NO in P-UAEC, which was not otherwise seen in
NP-UAEC. PGI2 production was similarly found in
P-UAEC in response to AII and ATP, but only in response to ATP in
NP-UAEC. Of the growth factors tested, maximally mitogenic doses of
bFGF, EGF, and VEGF all stimulated NO production in both NP- and P-UAEC
(Fig. 5A), but for
PGI2 biosynthesis (Fig. 5B), P-UAEC responded to
all three growth factors, whereas NP-UAEC only responded significantly
to VEGF.
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). Although a robust acute response was
observed after ATP challenge (10 µM), no response was
seen to AII (100 nM; Fig. 6A) or any of the growth factors
(10 ng/ml; Fig. 6B). Similar responses were seen in both P-UAEC and
NP-UAEC, with the increase in Ca2+ in NP-UAEC
being slightly less than that in P-UAEC. The effect of ATP on both
P-UAEC and NP-UAEC was blocked by suramine (not shown), suggesting a
receptor of the P2 subclass.
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),
significant increases in ERK-2 phosphorylation were seen in response to
all agonists tested. In NP-UAEC, however, there was a noticeable lack
of ERK-2 phosphorylation in response to ATP, AII, bFGF, and VEGF and
much impaired activation in response to TPA, suggesting reduced
coupling efficiency. This was further confirmed by the finding that
total ERK-1/2 was unaltered in NP-UAEC vs. P-UAEC, and that
EGF still evoked a strong activation of ERK-2 in NP-UAEC comparable to
that seen in P-UAEC.
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). Comparison of
NO production vs. PGI2 production for
P-UAEC shows that the relationship for growth factors vs.
nongrowth factors is clearly different, with growth factors all giving
noticeably more NO relative to PGI2 in response
to each agonist. This relationship is also clearly altered from that in
NP-UAEC. Comparison of PGI2 production
vs. ERK-2 phosphorylation is also of interest, as in P-UAEC
the results show that regardless of the agonist, there is a direct
relationship, whereas in NP-UAEC there is clearly no such relationship,
and EGF in particular stands alone in its ability to fail to stimulate
PGI2 production in the face of ERK-2
phosphorylation. Finally, comparison of NO production vs.
ERK-2 phosphorylation is striking in that the relationship for growth
factors is clearly different from that for nongrowth factors and is
strikingly reminiscent of that for NO per U PGI2
production, except with regard to EGF. In the case of EGF there is
comparatively strong ERK-2 phosphorylation with comparatively little NO
production relative to bFGF or VEGF. In the case of NP-UAEC, however,
for all agonists except EGF there is no apparent relationship between
NO production and ERK-2 phosphorylation, whereas for EGF stimulation
there is the same relationship as in P-UAEC.
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Discussion |
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Heptahelical receptor agonists (AII and ATP) are not the only factors that control vasodilator production. In addition to their angiogenic role, bFGF and VEGF have been implicated in control of uterine blood flow. Growth factor receptors are known to be expressed on uterine artery endothelium, which shows mitogenic responses to bFGF, EGF, and VEGF (7, 8). VEGF is known to be an important part of the regulation of normal pregnancy, in that it is an important mediator of the increases in angiogenesis as well as vascular permeability occurring at that time (19). In addition, VEGF in particular has recently been argued to be a key factor in the pathogenesis of preeclampsia (19), which is consistent with its known action to increase vascular permeability, a hallmark of preeclampsia. In addition, UA explants secrete PGI2 in response to growth factors in an endothelium-dependent manner (20). We have also shown directly the production of NO in response to growth factors in ovine fetoplacental artery endothelial cells (Zheng, J., personal communication).
Therefore, just how do growth factors and heptahelical receptor agonists achieve integrated control of vasodilator production in UA endothelium to maintain lower vascular resistance in the uteroplacental unit during normal pregnancy? To answer this question at the molecular level we require a cell model that until now has not been available. Herein we describe for the first time cell culture models derived from nonpregnant (NP-UAEC) and pregnant (P-UAEC) ewes that have retained not only the ability to produce both NO and PGI2 in response to a variety of agonists, but also show the same enhancement of AII- and ATP-induced vasodilator production in P-UAEC vs. NP-UAEC known to occur in vivo. Thus, we have been able to use these cell models to further investigate differences in the molecular integration of signaling responses to these different agonists in pregnancy with the integration (or lack of integration) in these same cells in the nonpregnant state.
The data from P-UAEC show that the relationship between the production of NO and PGI2 in response to heptahelical receptor agonists is divergent from that for growth factors. The importance of this finding is that it demonstrates the capacity for differential NO and PGI2 production by UAEC in response to each agonist, a feature also described previously in studies of bovine aorta endothelial cells (20A ). Furthermore, this finding suggests that such differential control of NO and PGI2 production is mediated at the level of alternate signal transduction pathway activation by each agonist. Although both heptahelical receptor agonists and growth factors can stimulate PGI2 production, both growth factors and AII failed to show mobilization of Ca2+, suggesting that at least some factors must work through alternate pathways. The additional findings that all agonists can activate ERK-1/2 and, further, that there is a direct relationship between the extent of ERK-2 phosphorylation and PGI2 production suggest that it is ERK-2, rather than Ca2+, that is the predominant physiological regulator of cPLA2 activation in P-UAEC.
The lack of a similar correlation for all agonists between NO production and ERK-2 phosphorylation suggests that the control of eNOS is not solely due to ERK-2, and the greater ability of the growth factors bFGF and VEGF to stimulate NO for a given parallel level of PGI2 production suggests that growth factors must be capable of stimulating additional signaling events that can potentiate NO production more fully than heptahelical receptors. As neither AII nor growth factors can stimulate Ca2+ mobilization in NP-UAEC, it follows that this difference does not relate to a Ca2+-based mechanism. Thus, alternate pathways are implied for signaling in response to growth factors, but not agonists of heptahelical receptors. Recent studies suggest that one possibility is protein kinase B, also known as AKT, activation (21, 22). An important exception to this observation is the data from EGF stimulation, which show some degree of agreement in the findings for correlation between ERK-2 phosphorylation and PGI2 production but relatively poor NO production relative to PGI2 production in comparison with bFGF and VEGF. The as yet unidentified pathway would be expected to be activated by bFGF and VEGF, but not EGF. This observation will be of value in the design of appropriate controls for future studies in this area.
With regard to the control of vasodilator production in NP-UAEC, the situation is clearly different from that in P-UAEC. The mitogenic responsiveness of these cells to growth factors is identical to that in P-UAEC, and the ability of agonists to evoke or not evoke Ca2+ mobilization is similar, if somewhat reduced, for ATP. The most striking difference at the level of signaling is the striking lack of coupling of agonist responses to ERK-2 phosphorylation, the important exception being the response to EGF. This highlights again the different responses to EGF, but also provides an important positive control for this experiment. In addition, we confirmed that altered coupling of other agonist responses to ERK-2 phosphorylation was not due to a lack of total ERK-1/2 protein and showed that this loss of coupling was also seen in response to TPA, confirming that it was not due to changes at the level of cell surface receptors. For heptahelical receptors, in the face of poor coupling to ERK-2, the rank order of PGI2 production (ATP>>AII) could be explained by the ability (or lack of ability) to mobilize Ca2+. However the lack of a potent effect of EGF on PGI2 production in NP-UAEC even in the face of ERK-2 phosphorylation suggests a more complicated scenario, whereby alteration of the coactivation of alternate pathways by an EGFR diminishes the necessity of ERK-1/2 for cPLA2 activation. Furthermore, a possible involvement of an alternate Ca2+-independent, ERK-1/2-independent pathway in stimulation of cPLA2 is demonstrated by the current finding that bFGF and VEGF can still stimulate PGI2 production in NP-UAEC without mobilizing Ca2+ or significantly stimulating ERK-2 phosphorylation.
Examination of similar data for NO production in NP-UAEC shows that the ability of a heptahelical receptor agonist to mobilize Ca2+ has no bearing on the activation of eNOS, as NO production is not seen in response to any of the agonists. The action of EGF in NP-UAEC on NO production is similar to that in P-UAEC, so the signaling pathways by which EGF activates eNOS (ERK-1/2 phosphorylation alone?) may be unaltered. The lack of ability of bFGF and VEGF to significantly activate ERK-1/2 phosphorylation or mobilize Ca2+ but still activate NO production suggests strongly that alternate pathways are responsible for eNOS activation in NP-UAEC.
In conclusion, we describe for the first time in UAEC that the dependence of NO production and PGI2 production on Ca2+ mobilization by agonists is less global than previously assumed, and that cell signaling through other pathways is a key determinant in the ability to differentially regulate vasodilator production through agonists using heptahelical receptors and growth factors. Furthermore, the pregnancy-induced refractoriness of the UA to vasoconstriction due to an increase in agonist-stimulated vasodilator production by uterine artery endothelium may be due to a marked alteration in coupling to alternate signaling pathways, which includes, but is not restricted to, activation of the ERK-1/2 signaling pathway. We believe that this is an important finding that impacts directly on our understanding of adaptation to healthy pregnancy and provides a model for investigation of abnormal conditions, such as intrauterine growth retardation and preeclampsia. In addition, this model provides us with a unique opportunity to further understand the molecular mechanisms that differentially regulate eNOS and cPLA2 activation in other cells.
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Footnotes |
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Received September 20, 1999.
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F.-X. Yi and I. M. Bird Pregnancy-Specific Modulatory Role of Mitochondria on Adenosine 5'-Triphosphate-Induced Cytosolic [Ca2+] Signaling in Uterine Artery Endothelial Cells Endocrinology, November 1, 2005; 146(11): 4844 - 4850. [Abstract] [Full Text] [PDF]
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 J. Zheng, Y. Wen, D.-b. Chen, I. M. Bird, and R. R. Magness Angiotensin II Elevates Nitric Oxide Synthase 3 Expression and Nitric Oxide Production Via a Mitogen-Activated Protein Kinase Cascade in Ovine Fetoplacental Artery Endothelial Cells Biol Reprod, June 1, 2005; 72(6): 1421 - 1428. [Abstract] [Full Text] [PDF]
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 K. A. Vonnahme, M. E. Wilson, Y. Li, H. L. Rupnow, T. M. Phernetton, S. P. Ford, and R. R. Magness Circulating levels of nitric oxide and vascular endothelial growth factor throughout ovine pregnancy J. Physiol., May 15, 2005; 565(1): 101 - 109. [Abstract] [Full Text] [PDF]
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J. M. Cale, S. C. Tsoi, M. Toppe, M. A. Grummer, M. Ochiai, R. R. Magness, and I. M. Bird Molecular Cloning of Ovine Endothelial Nitric Oxide Synthase and Expression in COS-7 Cells Reproductive Sciences, April 1, 2005; 12(3): 156 - 168. [Abstract] [PDF]
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C. M. Anderson, F. Lopez, H.-Y. Zhang, K. Pavlish, and J. N. Benoit Reduced Uteroplacental Perfusion Alters Uterine Arcuate Artery Function in the Pregnant Sprague-Dawley Rat Biol Reprod, March 1, 2005; 72(3): 762 - 766. [Abstract] [Full Text] [PDF]
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W. X. Liao, R. R. Magness, and D.-b. Chen Expression of Estrogen Receptors-{alpha} and -{beta} in the Pregnant Ovine Uterine Artery Endothelial Cells In Vivo and In Vitro Biol Reprod, March 1, 2005; 72(3): 530 - 537. [Abstract] [Full Text] [PDF]
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 F.-X. Yi, R. R. Magness, and I. M. Bird Simultaneous imaging of [Ca2+]i and intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R140 - R148. [Abstract] [Full Text] [PDF]
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D. Xiao and L. Zhang Adaptation of uterine artery thick- and thin-filament regulatory pathways to pregnancy Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H142 - H148. [Abstract] [Full Text] [PDF]
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K. A. Vonnahme and S. P. Ford Differential Expression of the Vascular Endothelial Growth Factor-Receptor System in the Gravid Uterus of Yorkshire and Meishan Pigs Biol Reprod, July 1, 2004; 71(1): 163 - 169. [Abstract] [Full Text] [PDF]
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Y. Li, J. Zheng, I. M. Bird, and R. R. Magness Mechanisms of Shear Stress-Induced Endothelial Nitric-Oxide Synthase Phosphorylation and Expression in Ovine Fetoplacental Artery Endothelial Cells Biol Reprod, March 1, 2004; 70(3): 785 - 796. [Abstract] [Full Text] [PDF]
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D.-b. Chen, I. M. Bird, J. Zheng, and R. R. Magness Membrane Estrogen Receptor-Dependent Extracellular Signal-Regulated Kinase Pathway Mediates Acute Activation of Endothelial Nitric Oxide Synthase by Estrogen in Uterine Artery Endothelial Cells Endocrinology, January 1, 2004; 145(1): 113 - 125. |
