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Endocrinology, doi:10.1210/en.2005-0399
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Endocrinology Vol. 147, No. 5 2442-2457
Copyright © 2006 by The Endocrine Society

Pregnancy-Enhanced Endothelial Nitric Oxide Synthase (eNOS) Activation in Uterine Artery Endothelial Cells Shows Altered Sensitivity to Ca2+, U0126, and Wortmannin But Not LY294002—Evidence that Pregnancy Adaptation of eNOS Activation Occurs at Multiple Levels of Cell Signaling

Jeremy A. Sullivan, Mary A. Grummer, Fu-Xian Yi and Ian M. Bird

Department of Obstetrics and Gynecology, Perinatal Research Laboratories, University of Wisconsin, Madison, Wisconsin 53715

Address all correspondence and requests for reprints to: Ian M. Bird, Ph.D., University of Wisconsin-Madison, Department of Obstetrics and Gynecology, Perinatal Research Laboratories, 7E Meriter Hospital/Park, 202 South Park Street, Madison, Wisconsin 53715. E-mail: Imbird{at}wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During pregnancy, vascular remodeling and vasoactive agents such as nitric oxide (NO) increase blood flow to the uteroplacental unit. Using our uterine artery endothelial cell (UAEC) culture model, based on cells from pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes, we investigate the relative physiological roles of Ca2+ vs. kinase in the regulation of endothelial NO synthase (eNOS) activity. When Ca2+ mobilization is fully inhibited using inhibitors of phospholipase C (PLC) (U73122) and the inositol triphosphate (IP3) receptor (IP3-R) (2-APB), significant residual eNOS activity remains in both P- and NP-UAEC. No change in ATP-stimulated ERK2, Akt, or eNOS phosphorylation is observed with U73122 (0.01–1 µM) or 2-APB (1–50 µM). The MAPK kinase (MEK) 1/2 inhibitor U0126 (10 µM) did not alter ATP-stimulated eNOS activity in P-UAEC, but potentiated the ATP response in NP-UAEC. Using two phosphatidylinositol 3-kinase (PI3-K) inhibitors, we observed no effect with LY294002 (10 µM) on eNOS activity in P- and NP-UAEC, but wortmannin (10 µM) inhibited both P- and NP-UAEC eNOS activation. Expression of constitutively active Akt (ca-Akt) in UAEC resulted in slight elevation of basal eNOS activity, but relative ATP-stimulated eNOS activation was not altered by ca-Akt. Wortmannin continued to inhibit eNOS activation by ATP in the presence of ca-Akt; LY294002 still had no inhibitory effect. Our data indicate both [Ca2+]i and multiple kinases are involved in the regulation of eNOS activity in our model. We report that pregnancy adaptation of eNOS activation includes the reduced sensitivity to ERK-mediated attenuation of eNOS activity and enhanced stimulation of eNOS activity through a wortmannin-sensitive, LY294002-insensitive, Akt-independent mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PREGNANCY IS A time of marked changes in the systemic and local uterine environment that facilitate the necessary increase in nutrients in the guise of blood flow to the uteroplacental unit (1, 2, 3, 4, 5). The increased nutrient demand is in part satiated by extensive remodeling of preexisting and new vasculature as well as the local endothelial production of effectors of vascular tone such as nitric oxide (NO) (4, 6). The up-regulation of endocrine factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiotensin II (AII) have been reported both in and around the uteroplacental unit (7, 8, 9). More recently, ATP has been implicated as a factor capable of increasing NO production in the uteroplacental unit during pregnancy (6, 10, 11, 12, 13, 14, 15).

In our model of uterine artery endothelial cells (UAEC) derived from both pregnant (P-UAEC) and nonpregnant ewes (NP-UAEC), we have previously reported a general pregnancy specific enhancement of coupling of physiologically relevant agonists such as VEGF, AII, and ATP to NO production as assessed by the measurement of NOx (total nitrate and nitrite converted to a reduced species) (12, 13). This pregnancy-specific enhancement is also associated with changes at the level of cell signaling as indicated by a general pregnancy enhanced coupling of factors to ERK2, a kinase often implicated in numerous cellular processes including endothelial NO synthase (eNOS) phosphorylation (16, 17, 18). In addition, a longer duration of the [Ca2+]i plateau is observed in both P-UAEC and vessels from pregnant animals as opposed to NP-UAEC and vessels from nonpregnant animals in response to ATP (14, 15).

In endothelial cells in general, activation of eNOS, the only detected isoform of NO synthase (NOS) in UAEC (19), has long been associated with increased Ca2+/calmodulin binding (20, 21, 22). It is believed that the Ca2+ increase is required to orientate eNOS into an active confirmation. More recently, it has been observed that NO may be produced even at basal levels of [Ca2+]i if eNOS is phosphorylated on specific residues (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32). Thus, the requirement for an increase in [Ca2+]i as a precursor to NO production may be supplanted by direct phosphorylation of eNOS. The lack of a general association between NOx production and Ca2+ mobilization in response to multiple agonists in UAEC implies a possible role for altered signaling in the endothelium during pregnancy including agonist-specific regulation of eNOS by kinases.

ATP, an agonist in our model that elicits both an increase in [Ca2+]i and ERK2 phosphorylation, is the prototypical agonist for us to investigate the extent to which Ca2+ mobilization or kinase activation control NO production in our UAEC model. We have previously reported the inhibition of ATP-stimulated NOx production with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA). Nonetheless, the use of BAPTA does not specifically inhibit agonist stimulated Ca2+ mobilization; rather, BAPTA indiscriminately sequesters Ca2+ to levels below basal, allowing us to conclude that eNOS is Ca2+ dependent, but the extent to which eNOS is [Ca2+]i sensitive in the physiological intracellular range remains unclear.

To further characterize the physiological relevance of the [Ca2+]i response after ATP treatment, we herein use two structurally and mechanistically dissimilar inhibitors to a characteristic PLC-stimulated, IP3-mediated Ca2+ mobilization, as would be inferred from our previously reported phosphoinositol turnover data and suramin inhibition studies (13). U73122, a PLC inhibitor and the structurally unrelated IP3-R antagonist, 2-APB, provide us with two separate and distinct means to inhibit Ca2+ mobilization without significantly perturbing basal [Ca2+]i levels, as occurs with BAPTA treatment. U73122 and 2-APB can both be used in our culture model to address the role Ca2+ mobilization plays not only in NO production and eNOS phosphorylation but also in ATP-stimulated ERK2 or Akt phosphorylation. The use of pharmacological inhibitors (U0126 and LY294002/ wortmannin) to prevent possible ERK2 or Akt phosphorylation also further delineates the possible role of kinase regulation of ATP-stimulated NO production. We report that, whereas eNOS activation by ATP is apparently [Ca2+]i sensitive, there is a discrepancy between the inhibition of [Ca2+]i response and eNOS activation in both NP-UAEC and P-UAEC. We further observe that multiple kinases appear to account for this discrepancy, and the relative roles of these kinases appear to be reprogrammed in pregnancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
U73122, 2-APB, and wortmannin were purchased from Calbiochem (San Diego, CA). LY294002 was purchased from Cell Signaling Technology Inc. (Beverly, MA). U0126 was purchased from Promega Corp. (Madison, WI). ATP (disodium salt) was purchased from Sigma-Aldrich Corp. (St. Louis, MO). Recombinant adenoviruses, Ad-CMV-Akt1 (Myr), and Ad-CMV-GFP were purchased from Vector Bioloabs (Philadelphia, PA). All cell culture reagents (liquid) were from Invitrogen (Carlsbad, CA) unless indicated and all tissue culture plasticware from Falcon (Fisher Scientific, Pittsburgh, PA).

Isolation of UAECs and general cell culture practice
Uterine arteries were obtained from Polypay and mixed Western breed nonpregnant sheep and pregnant ewes at 120–130 d of gestation during nonsurvival surgery, as described previously (12, 13). Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin-Madison Research Animal Care Committees of both the Medical School and the College of Agriculture and Life Sciences and follow the recommended AVMA guidelines for humane treatment and euthanasia of laboratory farm animals. Briefly, primary uterine arteries were flushed free of blood using M199 medium, before tying off arterial branches, clamping off the larger diameter end, and inflating with M199 containing 5 mg/ml collagenase B (Roche Molecular Biochemicals, Indianapolis, IN) and 0.5% BSA (Fraction V; Sigma) through a luerlock three-way tap. Digestion was allowed to proceed at 37 C for 55 min before flushing the collagenase solution and endothelial cell sheets from the inner surface of the vessel. Freshly isolated cells (passage 0) were plated to 35-mm dishes in MEM containing 20% FBS, 1% penicillin-streptomycin, 1% gentamycin (growth medium; used throughout). Cells were then grown and passaged as previously described over 12–16 d to approximately 70% confluence in T75 flasks at which point they were 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 approximately 70% confluence and subcultured for experimental use (passages 4–5).

Fura-2 [Ca2+]i imaging studies
Passage 4 UAEC were grown in 35-mm glass bottom microwell dishes (MatTek Corp., Ashland, MA) for 1–3 d before use in experiments. Cells were incubated in 5 µM Fura-2 AM (Molecular Probes Inc., Eugene, OR) with 0.05% Pluronic F127 (Molecular Probes Inc.) dissolved in Krebs buffer [125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM KH2PO4, 6 mM glucose, 2 mM CaCl2 ,25 mM HEPES (pH 7.4)] for 45 min at 37 C. The cells were washed with Krebs buffer, covered in 2 ml of Krebs buffer, and incubated for 30 min to allow complete ester hydrolysis. Cells were removed from the incubator, washed, and covered with 1 ml of Krebs buffer at room temperature. The dish was placed in the field of view and Fura-2 loading was verified by viewing at 380 nm UV excitation on a Nikon inverted microscope (InCyt Pm2; Intracellular Imaging, Inc., Cincinnati, OH). The cells were subsequently incubated with the appropriate agonist and/or antagonist, and the data were recorded for several individual cells using alternate excitation of 340 nm and 380 nm at 50-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 [Ca2+]i was calculated in real time using the InCyt Pm2 software. For all [Ca2+]i imaging experiments, data were recorded for 30 sec before agonist stimulation or antagonist addition to establish the basal [Ca2+]i.

eNOS activation assay
Cells were grown on T75 flasks to near confluency, at which time they were passaged onto 12-well plates and grown to 80% density. Cells were then washed twice and incubated for 1 h in Krebs buffer. [3H]Arginine (Amersham Biosciences, Piscataway, NJ; TRK 698, 1.5 µCi/well), and vehicle or agonist were subsequently added. Cells were incubated for 10–30 min as required. Reactions were stopped with ice cold 15% perchloric acid (final concentration 5%). [14C]Arginine (Amersham Biosciences; CFB 63, 100 nCi/well) was added for data normalization. Insoluble cell debris was removed with 5 min of centrifugation at 12,000 x g. Samples were extracted using a 1:1.25 volume of 1,1,2-trichlorotrifluoroethane/tri-n-octylamine (1:1 volume). The extracted samples were then applied to AG 50W-X8 cation-exchange resin (Bio-Rad Laboratories, Hercules, CA) (Na+ form), and citrulline flow through was collected using 25 mM HEPES (pH 5.5), 2 mM EDTA, and 2 mM EGTA. Arginine was eluted from the column with 50 mM KOH into a separate scintillation vial and subsequently neutralized by the addition of 400 µl of 1 M HEPES. InstaGel Plus scintillation fluid (PerkinElmer, Boston, MA) was added to each vial and the samples counted on the TRI-CARB 2300TR Liquid Scintillation Analyzer (PerkinElmer).

Phosphorylation-specific Western analyses
UAEC grown to 70% confluency on T75 flasks were passaged to 60-mm dishes and maintained for 24 h. After serum withdrawal/incubation for 4 h in 3 ml growth media without serum (containing 0.01% BSA), cells were stimulated with a dose of ATP (100 µM) observed to elicit maximal ERK2 phosphorylation in UAEC (12, 13) for the previously determined maximum stimulation time of 10 min (13). Antagonists were added 20 min before ATP treatment. 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 Na(PO4)2, 50 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM EDTA, 10 mM NaF, 2 mM Na3(VO4)2, 1 mM phenylmethylsulfonylfluoride, 1% Triton X-100, 5 µg/ml leupeptin, and 5 µg/ml aprotinin] before sonication, protein determination (bicinchoninic acid assay; Sigma), and Western blotting (20 µg per lane) on 7.5% polyacrylamide gels as described (12, 13). Phosphorylation of S1179,T497, S635, and S617 on eNOS was detected on separate membranes using commercially available phosphorylation-specific antibodies (eNOS S1179: Cell Signaling Technology, rabbit polyclonal no. 9571; 1:750/secondary (2°): Cell Signaling Technology, donkey antirabbit no. 7071, 1:3500; eNOS T497: Upstate, rabbit polyclonal no. 07185; 1:1000/2°: Amersham Biosciences, donkey antirabbit F(ab)2 NA9340,1:2000; eNOS S635: Upstate, rabbit polyclonal no. 26837; 1:5000/2°: Cell Signaling Technology, donkey antirabbit no. 7071, 1:3000; eNOS S617: Upstate, rabbit polyclonal no. 26836; 1:2500/2°: Cell Signaling Technology, donkey antirabbit no. 7071, 1:3000). Blots were frozen and blocked, after which phosphorylation of ERK2 (Y202/T204) and Akt (S473) were subsequently assessed using phosphorylation-specific antibodies to ERK2 (Promega AntiActive PMAPK rabbit polyclonal no. V8031; 1:2500/2°: Amersham Biosciences, donkey antirabbit F(ab)2 1:2500) and Akt [Cell Signaling Technology, rabbit polyclonal no. 9272; 1:750/2°: Amersham Biosciences, donkey antirabbit F(ab)2, 1:3000], and visualized using enhanced chemiluminescence plus (ECL+, Amersham Biosciences) reagents. To ensure consistent protein loading, all phosphorylation state-specific antibodies were normalized to total levels of ERK2 protein detected (Cell Signaling Technology, rabbit polyclonal no. 9101; 1:3000/2°: Cell Signaling Technology, donkey antirabbit no. 7071, 1:5000) or total levels of HSP90 (Affinity Bioreagents, rabbit polyclonal no. PA3-013; 1:20,000/2°: Amersham Biosciences, donkey antirabbit F(ab)2, 1:3000) where appropriate. Bands were visualized by use of an HP Deskscan system (Hewlett-Packard, Palo Alto, CA) and quantified using Molecular Analyst software (version 1.4; Bio-Rad Laboratories, Inc.).

Adenovirus infection
Cells were grown on T75 flasks to near confluency, at which time they were passaged onto six-well plates at 3.5 x 105 cells/well in 2 ml growth media. After 18 h, adenovirus [Ad-CMV-Akt1 (Myr) (ca-Akt) or Ad-CMV-GFP (green fluorescent protein, GFP)] was added to the media at a multiplicity of infection of 100, based on the number of cells plated the previous day. Preliminary experiments revealed almost 100% infection incorporation. A 24-h incubation with adenovirus was followed by the eNOS activity assay, described above, using 3 µCi [3H]arginine and 200 nCi [14C]arginine per well. The pellets remaining after the 12,000 x g centrifugation were immediately digested in 0.5 M NaOH for 30 min at 4 C. Protein levels were determined by the detergent-compatible protein assay method (Bio-Rad). In our preliminary studies Western analysis for GFP and phospho-Akt was performed on the digested pellets to ensure adenoviral protein expression was consistent. Phosphorylation-specific Western analysis experiments as described above were conducted in parallel six-well dishes after the 24-h adenovirus incubation to investigate the role adenovirus and/or wortmannin and LY294002 treatment had on the phosphorylation of eNOS in P- and NP-UAEC.

Statistical analyses
The data were analyzed by ANOVA or Student’s t test where appropriate. Results were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Assessment of eNOS activation and comparison with NOx measurement
Whereas we have previously used NOx accumulation as an indirect measurement of NO production (12, 13), we herein have implemented a modified arginine-citrulline activity assay (33) as an alternate and more direct measure of eNOS activity to confirm earlier results. Specifically, we wished to confirm that ATP, along with a number of growth factors and AII elicit an increase in NOx production that is generally greater in P-UAEC than NP-UAEC. The results for eNOS activity after treatment with ATP, VEGF, bFGF, epidermal growth factor (EGF), and AII are illustrated in Fig. 1AGo. Use of the activity assay indicated a statistically significant increase in activity in P-UAEC for ATP, VEGF, bFGF, and AII. No agonist other than ATP and VEGF used in this study invoked a significant increase in eNOS activity in NP-UAEC. The use of the arginine mimitec, L-NAME (100 µM) significantly decreased the ATP stimulation of eNOS activity, whereas the stereo-isomer D-NAME (100 µM) showed no significant inhibition (Fig. 1AGo, inset), strongly indicating a NOS-mediated product.


Figure 1
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  		FIG. 1. Assessment of eNOS activity in UAEC by arginine-citrulline conversion assay. A, Agonist stimulated eNOS activity in UAEC. Multiple agonists were examined to assess their ability to stimulate eNOS activity using an arginine-citrulline conversion assay. ATP (100 µM), VEGF, bFGF, EGF (10 {eta}g/ml), and AII (0.1 µM) were all given as 30-min treatments, and activity was assessed as described in Materials and Methods. Values shown are mean ± SE of n = 4 independent experiments (*, P < 0.05 relative to control; #, P < 0.05 vs. same P-UAEC response). Inset, ATP-stimulated activity was measured in the presence of L-NAME (100 µM) and D-NAME (100 µM) (#, P < 0.05 relative to ATP treatment). B, Dose dependency of eNOS activity after ATP (1–300 µM) treatment in P-UAEC and NP-UAEC (bar). Values shown are mean ± SE of n = 4 independent experiments each (*, P < 0.05 vs. same UAEC control; #, P < 0.05 vs. same P-UAEC response). C, Comparison of NO detection using the Sievers NO Analyzer (NOA 280) and the arginine-citrulline conversion assay. Data collected from both the arginine-citrulline conversion assay and the NOA 280 were expressed as a percentage of the mean of 100 µM ATP. Error bars represent variation in the conversion assay (vertical bars) and the NOA 280 measurement of NOx (horizontal bars).

 
In agreement with our previous studies, the eNOS activity assay with ATP treatment in P-UAEC exhibited a dose-dependent trend of eNOS activation; a similar but smaller magnitude trend was also apparent in NP-UAEC (Fig. 1BGo). Expressing our former NOx data and the current eNOS activity assay data as a percentage mean of the maximum stimulatory dose of ATP (100 µM) demonstrated a close correlation between the assays (Fig. 1CGo).

Effect of 2-APB and U73122 on ATP-stimulated Ca2+ mobilization and eNOS activity
To further characterize the role that increases in [Ca2+]i from basal levels play in eNOS activation, we employed two structurally dissimilar compounds to block agonist-induced Ca2+ mobilization, 2-APB and U73122. Full-field [Ca2+]i data were collected as both a peak value, that being the initial spike upon agonist stimulation, as well as a sustained phase of Ca2+ mobilization, or subsequent plateau, as determined in this study as the maximum measured plateau value achieved during a 5-min stimulation. Selective inhibition of the IP3-R with 2-APB in P-UAEC exhibited an IC50 value for peak [Ca2+]i inhibition at 8 µM, whereas the observed plateau IC50 value was 1.0 µM (Fig. 2Go, top). In NP-UAEC, the IC50 for peak [Ca2+]i inhibition with 2-APB was 6 µM, whereas the plateau value IC50 was too low to measure. The observed IC50 values for 2-APB inhibition of eNOS activity were 30 µM in P-UAEC and 20 µM in NP-UAEC, both values well above the observed IC50 in P- and NP-UAEC peak and plateau [Ca2+]i measurements.


Figure 2
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  		FIG. 2. Effects of IP3-R and PLC antagonists on the ATP-stimulated increase in [Ca2+]i vs. eNOS activation. P- and NP-UAEC were treated with ATP (100 µM) for 10 min with or without 5 min pretreatment with 2-APB (top) or U73122 (bottom) at the indicated doses to assess any changes in [Ca2+]i using Fura-2 imaging and changes in eNOS activity using an arginine-citrulline conversion assay. Alterations in [Ca2+]i levels after treatment were assessed as both change in peak and plateau [Ca2+]i levels and expressed as the percentage of ATP response alone (n = 4–6). Parallel experiments investigating eNOS activity as outlined in Materials and Methods were also performed and are similarly expressed as a percentage of ATP response alone (n = 4). Values shown are mean ± SE (*, P < 0.05 relative to ATP response).

 
After determination of the effects of 2-APB in our UAEC model, we next addressed the effects of U73122. The reasonably selective phosphoinositide-specific PLC antagonist U73122 showed significant inhibition of ATP-stimulated [Ca2+]i increase (Fig. 2Go, bottom). In P-UAEC, the observed IC50 for U73122 [Ca2+]i inhibition was 0.05 µM for peak [Ca2+]i values and 0.02 µM for plateau values. In NP-UAEC, the peak and plateau values were of the same order of magnitude at 0.09 µM and 0.07 µM respectively. The observed IC50 values for U73122 inhibition of ATP-stimulated eNOS activity were again higher than that observed in both peak and plateau [Ca2+]i inhibition at 1 µM and 0.8 µM in P- and NP-UAEC, respectively.

Investigation of Ca2+ mobilization inhibitors on ERK2, Akt, and eNOS phosphorylation
The fact that ATP exhibits a pregnancy enhanced coupling to ERK 2 in our model, along with pregnancy enhanced eNOS activation prompted us to investigate whether the ATP coupling to ERK2 activation was inhibited by the previously employed inhibitors of agonist stimulated [Ca2+]i increase. Similar to previous results (12, 13), phosphorylation of ERK2 in response to ATP in this study was significantly increased 2.17-fold in P-UAEC; but the 1.14-fold increase in NP-UAEC was not significant (data not shown). Figure 3Go represents quantified phospho-specific Western analyses of ATP-stimulated ERK2 and Akt phosphorylation in the presence of 2-APB (1–50 µM) or U73122 (0.01–10 µM) in P- and NP-UAEC. Pretreatment with 2-APB (bottom left) or U73122 (bottom right) resulted in no significant decrease in ERK2 phosphorylation in both P- and NP-UAEC at any of the doses that inhibited [Ca2+]i or eNOS activity (Fig. 2Go). The investigation of Akt phosphorylation demonstrated no detectable change with ATP stimulation (data not shown), also as previously demonstrated (12, 13), and 2-APB (top left) or U73122 (top right) pretreatment at doses that effectively blocked Ca2+ mobilization or eNOS activity had no effect on Akt phosphorylation (Fig. 3Go).


Figure 3
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  		FIG. 3. ATP-stimulated Akt and ERK2 phosphorylation (P-Akt and P-ERK2) in UAEC with 2-APB or U73122 pretreatment. After serum withdrawal, P- and NP-UAEC were treated with ATP (100 µM) for 10 min as described in Materials and Methods with or without a 10-min pretreatment of 2-APB (left) or U73122 (right) at the indicated concentrations. Phospho-specific Western analysis was performed as described in Materials and Methods. Representative Western blots are presented above. Graphs represent mean ± SE of n = 4 independent experiments with data normalized to total corresponding ERK2 protein and expressed as the percentage of ATP response (*, P < 0.05 relative to control).

 
In light of reports that kinases other than ERK2 or Akt may be involved in eNOS activation through altered phosphorylation of known sites on eNOS (33, 34, 35, 36), we examined possible changes in four proposed regulatory phosphorylation sites (S1179, S635, S617, and T497) on eNOS using phospho-specific Western analyses after ATP treatment (100 µM). Figure 4Go indicates that in P-UAEC, ATP treatment stimulated a time-dependent, significant increase in S1179, S635, and S617 phosphorylation within 10 min, consistent with our previous results (33). We further show herein that, although there was a similar weaker trend observed in the response of NP-UAEC, there was no significant increase in eNOS phosphorylation over time in NP-UAEC with ATP treatment.


Figure 4
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  		FIG. 4. Time-course of ATP-stimulated eNOS phosphorylation in UAEC. UAEC were treated with ATP (100 µM) for the indicated times and phospho-specific Western analysis for four different phosphorylation sites on eNOS in both P- and NP-UAEC (S1179, T497, S617, and S635) was performed. Graphs represent mean ± SE of n = 4–6 independent experiments with data normalized to total corresponding HSP90 protein and expressed as fold of 0 time control (*, P < 0.05 relative to control).

 
To eliminate a possible indirect role of IP3-mediated Ca2+ release in the phosphorylation of eNOS, we examined the effect of 2-APB on ATP-stimulated eNOS phosphorylation (Fig. 5Go). No alteration of ATP-stimulated eNOS phosphorylation at any of the four phosphorylation sites was observed at any tested concentration of 2-APB (1, 10, and 50 µM). Similarly, when we examined the effect of U73122 on ATP-stimulated eNOS phosphorylation (Fig. 6Go), no significant alteration of ATP-stimulated eNOS phosphorylation occurred at any dose tested (0.01, 1, and 10 µM).


Figure 5
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  		FIG. 5. Effects of 2-APB on phosphorylation of eNOS. Phospho-specific Western analysis using antibodies to four phosphorylation sites on eNOS (P-eNOS) (S1179, T497, S617, and S635) was performed after a 10-min ATP (100 µM) treatment in the presence of 2-APB (1, 10, 50 µM; 10 min pretreatment) in P- and NP-UAEC. Representative Western blots are presented above. Graphs represent mean ± SE of n = 4–6 independent experiments with data normalized to total corresponding HSP90 protein and expressed as a percentage of ATP response without inhibitor (*, P < 0.05 relative to control).

 

Figure 6
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  		FIG. 6. Effects of U73122 on phosphorylation of eNOS. Phospho-specific Western analysis using antibodies to four phosphorylation sites on eNOS (S1179, T497, S617, and S635) was performed with ATP (100 µM, 10 min) treatment in the presence of U73122 (0.01, 1, 10 µM; 10 min pretreatment) in P- and NP-UAEC. UAEC were treated and Western analysis was performed as indicated in Materials and Methods under the same conditions outlined in Fig. 3Go. Representative Western blots are presented above. Graphs represent mean ± SE of n = 4–6 independent experiments with data normalized to total corresponding HSP90 protein and expressed as a percentage of ATP response without inhibitor (*, P < 0.05 relative to control).

 
Effect of U0126, LY294002, and wortmannin on eNOS activity
Using inhibitors to the ERK 2 (U0126) and PI3-K/Akt (LY294002, wortmannin) cascades to implicate either signaling cascade’s involvement in the ATP-stimulated increase in eNOS activation, we observed that the MEK 1/2 inhibitor U0126 did not affect ATP-stimulated eNOS activation in P-UAEC (Fig. 7Go). Furthermore, pretreatment of NP-UAEC with U0126 had a stimulatory effect on ATP activation of eNOS, raising the levels of eNOS activation to that seen in P-UAEC. Conversely, the PI3-K inhibitor LY294002 (10 µM), did not decrease ATP-stimulated eNOS activity. The structurally and mechanistically dissimilar inhibitor of PI3-K, wortmannin (10 µM), however, exhibited a statistically significant decrease in ATP-stimulated eNOS activation in both P- and NP-UAEC, lowering the levels of eNOS activity to a common depressed level that was still above basal. None of the inhibitors caused any significant alteration in basal eNOS activity.


Figure 7
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  		FIG. 7. Inhibitors of the MAPK and PI3-K signaling cascades on ATP-stimulated eNOS activity. ATP (100 µM, 30 min) stimulated eNOS activity assessed by the arginine-citrulline conversion assay outlined in Materials and Methods was performed in NP- and P-UAEC with or without a 20-min pretreatment of a MEK 1/2 inhibitor U0126 (10 µM), or two selective, structurally dissimilar PI3-K inhibitors, LY294002 (LY; 10 µM) and wortmannin (WORT; 10 µM). Graph represents mean ± SE of n = 4 separate experiments with data expressed as fold of control response (*, P < 0.05 relative to control; #, P < 0.05 relative to respective ATP response; +, P < 0.05 relative to respective NP response). Cont, Control.

 
Effect of ca-Akt alone or with LY294002 or wortmannin on eNOS activity
As an alternate and independent approach to further evaluating the role of the PI3-K/Akt pathway and the specificity of LY294002 and wortmannin as inhibitors of eNOS activation, we also used adenoviral transfer and expression of ca-Akt in UAEC. Infection with GFP (as viral control) and ca-Akt was readily verified by Western analyses of phospho-Akt and GFP protein expression (Fig. 8AGo). Although phosphorylated ca-Akt was detectable at considerably higher levels than in control cells, verifying activation had occurred, the actual amount of total Akt protein was still just over 2-fold of control in ca-Akt infected cells with respect to control and GFP infected cells (Fig. 8AGo). GFP caused a negligible change in basal eNOS activation, suggesting the adenovirus vehicle alone was not impacting significantly on the system under examination. Cell treatment with ca-Akt alone caused a small observable increase in eNOS activity, but this was not significant (data not shown). Furthermore, the presence of ca-Akt (or GFP) did not alter the magnitude of any further ATP-stimulated increase in eNOS activity in NP- or P-UAEC (Fig. 8BGo). In addition, consistent with the data in Fig. 7Go, LY294002 (10 µM) did not in turn affect the ATP-stimulated increase in eNOS activity in GFP or ca-Akt infected UAECs (Fig. 8CGo), but wortmannin (10 µM) continued to significantly decrease ATP-stimulated activity in the presence of both GFP and ca-Akt.


Figure 8
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  		FIG. 8. Effect of adenovirus and PI3-K inhibitors on ATP-stimulated eNOS activity. A, Expression of adenovirus-infected proteins. P- and NP-UAEC were infected with adenoviruses Ad-CMV-GFP or Ad-CMV-Akt1 (Myr), followed by 24-h incubation. Representative Western blot of samples revealed phospho-Akt (P-Akt) (Ser 473), and GFP was readily detected after infection with the respective adenovirus at the expected molecular weights. B, Adenovirus effect on eNOS activation. ATP (100 µM, 10 min)-stimulated eNOS activity was assessed by the arginine-citrulline conversion assay as outlined in Materials and Methods in NP- and P-UAEC. Data are expressed as a percentage of control (no adenovirus) ATP response, after normalizing for basal activity for each adenovirus. Graph represents mean ± SE of n = 5 separate experiments. C, Inhibitor effects on eNOS activation. Experiments performed as above, with or without a 20-min pretreatment of LY294002 (LY; 10 µM) or wortmannin (WORT; 10 µM). Graph represents mean ± SE of n = 5 separate experiments with data expressed as a percentage of same adenovirus ATP response (*, P < 0.05 relative to control ATP response). Cont, Control.

 
Effect of ca-Akt and LY294002 or wortmannin on eNOS phosphorylation
In view of the finding that ca-Akt could not alter the effects of LY294002/wortmannin on eNOS activity, we further investigated the parallel effects of ca-Akt alone or combined with these inhibitors on eNOS phosphorylation. Phospho-specific Western analysis revealed that basal phosphorylation of S1179, T497, S617, and S635 were unaffected upon infection with either GFP or ca-Akt in both P- and NP-UAEC. In GFP and ca-Akt infected P-UAEC, ATP-stimulated significant phosphorylation of S1179, S617, and S635 consistent to results reported in Fig. 4Go; furthermore, there was no significant increase in T497 phosphorylation. Basal phosphorylation of S1179, S617, and S635 was inhibited in control P-UAEC by LY294002 and wortmannin (data not shown). Basal levels of S1179, T497, S617, and S635 in NP-UAEC were observed to change only slightly with LY294002 and wortmannin, and only S1179 and S635 levels were significantly affected (data not shown). The ATP-stimulated increase of S617 eNOS phosphorylation was inhibited by LY294002 in both control and GFP infected P-UAEC, but not in ca-Akt infected P-UAEC. A similar, but nonsignificant, trend of S617 phosphorylation inhibition with wortmannin in both control and GFP infected P-UAEC was observed (see Fig. 10Go). There were no other effects of wortmannin or LY294002 on ATP-stimulated eNOS phosphorylation in P-UAEC. As observed previously in NP-UAEC (Fig. 4Go), ATP treatment did not significantly alter any of the four phosphorylation sites we investigated in control, GFP or ca-Akt infected cells (Figs. 9Go and 10Go). In the presence of wortmannin or LY294002, there was no observed change in phosphorylation at any of the four examined sites.


Figure 10
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  		FIG. 10. Effect of adenovirus and PI3-K inhibitors on ATP-stimulated S617 and S635 eNOS phosphorylation. NP- and P-UAEC were infected with adenoviruses Ad-CMV-GFP or Ad-CMV-Akt1 (Myr), followed by 24-h incubation. ATP (100 µM, 10 min) stimulated S617 (top) and S635 (bottom) eNOS phosphorylation with or without a 20-min pretreatment of the PI3-K inhibitors, LY294002 (LY; 10 µM) or wortmannin (WORT; 10 µM) was assessed using phospho-specific Western analysis as described in Materials and Methods. Graphs represent mean ± SE of n = 4 separate experiments, with data expressed as a percentage of same adenovirus ATP response. C, Control.

 

Figure 9
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  		FIG. 9. Effect of adenovirus and PI3-K inhibitors on ATP-stimulated S1179 and T497 eNOS phosphorylation. NP- and P-UAEC were infected with adenoviruses Ad-CMV-GFP or Ad-CMV-Akt1 (Myr), followed by 24-h incubation. ATP (100 µM, 10 min) stimulated S1179 (top) and T497 (bottom) eNOS phosphorylation with or without a 20-min pretreatment of LY294002 (LY; 10 µM) or wortmannin (WORT; 10 µM) was assessed using phospho-specific Western analysis as described in Materials and Methods. Data are expressed as a percentage of respective ATP response. Graphs represent mean ± SE of n = 4 separate experiments. C, Control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Paramount to our investigation of the pregnancy enhanced endothelial production of NO is an accurate and reproducible method to measure NO production. Our previous reports involving NO production relied on the use of the Sievers NO Analyzer (Sievers Instruments, Inc., Boulder, CO), which essentially converts total nitrate and nitrite in the culture media to a detectable species, termed NOx (37, 38, 39, 40). In our model, where eNOS is the predominate isoform of NOS, there is very low yield of product; any contaminating nitrate or nitrite makes detecting the small increases in NOx production that much more difficult (41). In light of this drawback, we decided to address NO production by investigating eNOS activity, measuring the radiolabeled citrulline produced after incubation with [3H]arginine. Comparing the two assays, we observe general agreement between them, the only exception being the loss of the small EGF response in P-UAEC (Fig. 1AGo). The dose-dependent trend in ATP-stimulated NOx accumulation previously described (13) was similarly observed using the activity assay (Fig. 1BGo). We also observed selective inhibition of the ATP response by L-NAME, as would be expected with a NOS mediated event (Fig. 1AGo, inset). Comparing the NOx and the eNOS activity data, there is reasonable agreement between the assays; the greatest variation in the measurements, horizontal error bars vs. vertical error bars, is apparently due to the difficulty in measuring lower levels of NOx, indicating that more precise measurements of low activity can be made with the radioactive assay (Fig. 1CGo).

A fundamental role for [Ca2+]i activation of eNOS in UAEC was previously implicated through the obliteration of ATP-stimulated NOx production in the presence of BAPTA (13). To investigate whether eNOS activation in UAEC is actually sensitive to [Ca2+]i, we chose to monitor changes in ATP-stimulated eNOS activity as the ATP-stimulated increase in [Ca2+]i was progressively blocked. In our current studies, we employed two chemically and mechanistically dissimilar inhibitors of agonist stimulated [Ca2+]i increase, 2-APB (42, 43, 44) and U73122 (45, 46, 47, 48), both inhibitors addressing a classical P2Y-mediated ATP-stimulated [Ca2+]i increase at the level of generation of IP3 (U73122) and at the IP3-R itself (2-APB). Interestingly, the IP3-R antagonist, 2-APB, exhibits an IC50 almost eight times lower for plateau inhibition of the [Ca2+]i response as opposed to peak inhibition in P-UAEC. This suggests either different IP3-R isoforms may mediate acute vs. sustained phases of ATP-stimulated Ca2+ mobilization, or IP3-R modification occurs through phosphorylation or binding of other proteins accounting for the discrepancy. In NP-UAEC, 2-APB resulted in a similar IC50 for peak inhibition as observed in P-UAEC, but the plateau [Ca2+]i response in NP-UAEC was too low above basal to detect in our system, making 2-APB capable of abolishing the NP-UAEC [Ca2+]i plateau. The results we observe for inhibition of Ca2+ mobilization with 2-APB in our model are consistent with that reported by others (42, 43). The use of 2-APB as a tool for investigating the role of IP3-R has come into question by some groups that observe that 2-APB may inhibit SERCA [sarco(endo)plasmic reticulum Ca2+-ATPase] pumps (49, 50), or other nonspecific Ca2+ signaling events (51, 52). Even at extremely high concentrations of 2-APB alone (50 µM or above), we do not observe an influx of Ca2+ otherwise reported by some groups, nor do we observe morphological changes after pretreatment (data not shown). Furthermore, general agreement between the effects of 2-APB and U73122 supports the involvement of IP3 generation in both the initial ATP-stimulated [Ca2+]i response and indicates IP3 generation is further required throughout the sustained phase. Such observations strongly indicate a role for IP3-R in regulating sustained Ca2+ influx across the plasma membrane as well as initial intracellular Ca2+ release. The doses of U73122 used in our studies are consistent with those used by other groups in various cell types (46, 48, 53, 54). Figure 2Go also illustrates the alteration in eNOS activity with 2-APB and U73122 pretreatment with ATP stimulation, revealing that in all cases examined in our model, the observed decline in eNOS activity occurs at higher concentrations of both 2-APB and U73122 than those required to decrease peak or plateau [Ca2+]i levels. This supports the notion that eNOS is sensitive to [Ca2+]i through the physiological range, but this is not the sole mechanism regulating eNOS activity upon ATP stimulation in our model and that there is an additional component involved in the ATP stimulation of eNOS activity.

The unknown, [Ca2+]i-independent component of ATP-stimulated eNOS activation has been implicated by studies of eNOS in many endothelial cell types to be the result of kinase phosphorylation of eNOS, rendering it active at basal [Ca2+]i levels. Two of many candidate kinases possibly involved in the phosphorylation of eNOS are ERK2 and Akt, both of which are present and undergo agonist-specific activation in our model (12, 13, 14, 15). We have previously reported a pregnancy-enhanced activation of ERK2 by ATP, and also that ATP receptors do not efficiently couple to Akt phosphorylation (12, 13). Although there has been no formal clarification of the [Ca2+]i sensitivity of either ERK2 or Akt, we observed no significant change in ATP-stimulated phosphorylation of ERK2 nor any observable change in phosphorylated Akt with either 2-APB or U73122 at the doses shown (Fig. 3Go), indicating that either kinase may be a candidate for the 2-APB/U73122-insensitive component of eNOS activation.

There are numerous suggested phosphorylation sites on eNOS that may confer regulation, and although an exhaustive investigation of site-directed mutants is warranted and was recently undertaken on ovine eNOS expressed in COS-7 cells (55), we have made use herein of phospho-specific Western analysis in investigating ATP-stimulated eNOS phosphorylation in UAEC at four of the more well-described potential phosphorylation sites, S1179, T497, S617, and S635. Although there is no absolute relationship between activation or inhibition of eNOS when phosphorylation is present on any of the aforementioned residues, the relative changes in the phosphorylation state of eNOS may provide insight into pregnancy-specific changes in cell signaling and their impact on the enzyme’s structure/function. Time-course data for ATP-stimulated eNOS phosphorylation show that ATP stimulates a significant increase in S1179, S617, and S635 phosphorylation in P-UAEC at or less than 10-min treatment (Fig. 4Go), which is entirely consistent with previous observations in our P-UAEC model (33). In NP-UAEC, we extend our previous findings to show no significant phosphorylation at any of the examined time points. Thus, it seems overall that phosphorylation events are up-regulated in pregnancy, consistent with altered kinase signaling, although their significance remains unclear. Whereas ATP increases S1179, S617, and S635 eNOS phosphorylation in P-UAEC, neither 2-APB (Fig. 5Go) nor U73122 (Fig. 6Go) significantly alters the agonist stimulated phosphorylation of eNOS at any of the four examined sites, indicating phosphorylation is neither Ca2+-dependent nor [Ca2+]i-sensitive and that effects of 2-APB and U73122 on eNOS activity are not solely regulated by either S1179, S617, or S635 phosphorylation.

Our finding that the MEK 1/2 inhibitor U0126 did not decrease the ATP-stimulated increase in eNOS activity in P-UAEC, whereas in NP-UAEC, U0126 pretreatment actually significantly increased eNOS activity levels to that observed in P-UAEC was intriguing. This suggests one mechanistic basis for pregnancy adaptation may involve a MEK 1/2-sensitive, NP-UAEC-specific, negative regulator of eNOS activity. It is not yet clear whether this is mediated by direct or indirect phosphorylation, but our data are similar to that of Bernier et al. in bovine artery endothelial cells (16). In addition to our studies of the MEK/ERK pathway, the effect of the PI3-K inhibitor LY294002, an upstream inhibitor of Akt, was also examined in light of the findings of a role for Akt phosphorylation of eNOS in other endothelial models. In UAEC, we observed no statistically significant decrease in eNOS activation with LY294002 treatment, consistent with the lack of effect of ATP stimulation on Akt phosphorylation (12, 13). However, another commonly used inhibitor of PI3-K, wortmannin, did exhibit a statistically significant decrease in eNOS activation in both P- and NP-UAEC after ATP treatment and brought activity in P- and NP-UAEC down to a common level. The magnitude of this wortmannin-sensitive portion of eNOS activation could account for the greater portion of eNOS activation remaining in P-UAEC compared with NP-UAEC after [Ca2+]i elevation was fully inhibited (Fig. 2Go). Thus, it is possible the wortmannin-sensitive factor is common to both P- and NP-UAEC, but the extent to which it is involved in eNOS activation is greater in pregnancy.

Wortmannin has been shown to have a number of effects in various cell types including alterations in both [Ca2+]i and kinase signaling. Wortmannin may affect [Ca2+]i levels by altering activity of PLC{gamma}1 (56, 57), inhibiting agonist-stimulated IP3 levels (57), inhibiting other PI kinases such as PI4-Ks (59, 60) or by inhibiting store operated Ca2+ channels as has been observed in platelets (61). It would be tempting to conclude that the aforementioned effects associated with wortmannin by other groups were present in UAEC, but this seems unlikely because there is only a weak inhibitory effect on ATP-stimulated Ca2+ mobilization and higher doses of LY294002 known to inhibit PI4-K are also ineffective on ATP-stimulated eNOS activity (Sullivan, J. A., and M. A. Grummer, unpublished data). Nonetheless, wortmannin has also been shown in other cells to inhibit other kinases at a similar or lower IC50 to the dose used in this study, including Polo-like kinases (62) and myosin light chain kinases (63). Therefore, the results observed with wortmannin on eNOS activity in our model had to be further qualified. In particular, we wanted to ensure that Akt does not have a more subtle effect on eNOS activity and also further investigate any role PI3-K may play in ATP-stimulated eNOS activity independent of Akt. To achieve this, we had to uncouple Akt activation from the PI3-kinase pathway, which we did by introducing a constitutively active form of Akt (ca-Akt) into our model (Fig. 8Go). In light of our observations that ATP-stimulated UAEC do not show an increase in Akt phosphorylation, our phospho-specific Western analysis of Akt would be sufficient to detect this mutant Akt because it is believed to be readily phosphorylated once it is expressed. Theoretically, the mutant we employed would circumvent the actions of PI3-K through the Akt pathway and therefore any stimulatory effect would become insensitive to specific PI3-K inhibitors. Alternatively, any effect of ATP via PI3-K independent of Akt would be both LY294002 and wortmannin sensitive and any effect of ATP through other kinases would remain sensitive to wortmannin alone.

Increased elevation of Akt via infection with ca-Akt resulted in a corresponding incremental increase in basal eNOS activity in some but not all experiments, but it should be noted that despite substantial increases in phosphorylated Akt, the magnitude of this increase in basal eNOS activity was modest at best, and insignificant compared with the effects of known physiological/receptor-mediated stimuli such as ATP. The relatively weak eNOS activation observed with expression of ca-Akt was consistent with our previous finding that EGF, a potent stimulator of Akt phosphorylation in UAEC, does not promote a significant increase in eNOS activity (12). In addition, the response to ATP above basal in untreated cells, in cells expressing GFP, and in those expressing ca-Akt remained consistent (Fig. 8BGo), implying a lack of direct involvement of Akt in ATP-stimulated eNOS activity in our model. We conclude, therefore, that there is no fundamental role for Akt in ATP-stimulated eNOS phosphorylation and activation in the UAEC model, in contrast to the findings of some groups working in other endothelial cell types or with other stimuli (23, 24, 25, 26, 27, 28).

In addition to examining the effects of ca-Akt alone on basal and ATP-stimulated eNOS activity our finding that wortmannin was equally capable of inhibiting eNOS activation in response to ATP in control, GFP and ca-Akt infected cells, whereas LY294002 remained unable to inhibit ATP-stimulated eNOS activity in control, GFP or ca-Akt-infected cells allows us to conclude that the action of wortmannin is indeed by inhibition of another target that is not PI3-K and is not LY294002 sensitive.

With regard to the relationship between eNOS phosphorylation and pregnancy-specific eNOS activation, our parallel investigation of the effects of LY294002 and wortmannin on phosphorylation of eNOS revealed that only LY294002 was sufficient to inhibit ATP-stimulated eNOS phosphorylation, and this inhibition occurred only at S617. LY294002 inhibition of S617 phosphorylation in P-UAEC was observed in both control and GFP infected cells; however, in the ca-Akt-infected P-UAEC the LY294002 inhibition of S617 was lost. This observation does lead us to believe S617 is an LY294002-sensitive site, and therefore a plausible site of PI3-K/Akt regulation. Nonetheless, in light of the lack of inhibitory effect of LY294002 on eNOS activity in control and ca-Akt infected UAEC, the physiological relevance of phosphorylation at this residue in our model does not appear germane to eNOS activation.

Our findings regarding a dissociation of eNOS phosphorylation and activation responses are intriguing because one of the sentinel observations in other endothelial cells was an Akt-mediated phosphorylation of eNOS at S1179 (25, 26, 27, 28), rendering the enzyme active. In our UAEC model, there is a nonsignificant trend toward inhibition with wortmannin at S1179 in both control and GFP infected P-UAEC, and this trend was ameliorated in the ca-Akt-infected cells. It is possible that basal levels of phospho-Akt are involved in the routine cycle of phosphorylation/dephosphorylation of eNOS at sites such as S1179. We observed in nonstimulated cells that LY294002 and wortmannin were sufficient to inhibit basal phosphorylation of Akt, meaning the introduction of Akt protein, and even more so the infection with ca-Akt may provide more active Akt protein sufficient to maintain the basal level of eNOS phosphorylated at S1179 in the presence of inhibitors. Nonetheless, our activity data suggest again that this is not an important event for activation in response to agonists.

In summary, in both P- and NP-UAEC, eNOS activation is to some degree [Ca2+]i sensitive. However, the peak and plateau [Ca2+]i levels were reduced by inhibitors at concentrations much lower than those required to affect eNOS activity. The contribution of ERK2 in attenuating ATP stimulation of eNOS activity has yet to be completely defined. It is now clear that there is no role for the direct actions of Akt in ATP-stimulated eNOS activity in our model, as indicated by the lack of stimulated phosphorylation of Akt with ATP treatment, the lack of effect of ca-Akt on ATP stimulation of eNOS activity and the lack of effect of LY294002 in blocking ATP-stimulated eNOS activity in the absence or presence of ca-Akt. Examination of four of the more well-characterized phosphorylation sites, S1179, T497, S617, and S635 in both P- and NP-UAEC has indicated that, whereas changes in phosphorylation are enhanced during pregnancy, monitoring of these four sites alone may not be a suitable method for inferring activity. Examination of individual phosphorylation events has shown that, whereas S617 and to some extent S1179 phosphorylation of eNOS may indeed be PI3-K sensitive, any role this may play appears to be independent of agonist-stimulated eNOS activity in NP-UAEC or its enhancement in P-UAEC.

In our investigation of eNOS activation, the use of kinase inhibitors further indicated a potentiation of ATP-stimulated eNOS activity with the MEK inhibitor U0126 in NP-UAEC, whereas in P-UAEC there was no change. There is an apparent discrepancy observed with LY294002 and wortmannin, wherein LY294002 is ineffective at inhibiting eNOS activation and wortmannin inhibits both NP- and P-UAEC eNOS activity. The portion of eNOS activity that wortmannin inhibited may account for the greater residual eNOS activity remaining in P-UAEC over NP-UAEC when [Ca2+]i was fully inhibited with 2-APB. As such, the wortmannin-sensitive factor may be a plausible pregnancy-enhanced regulator of eNOS activity underlying pregnancy adaptation. Furthermore, the action of wortmannin appears independently of PI3-K and/or associated Akt because introduction of ca-Akt does not prevent wortmannin inhibition of ATP-stimulated eNOS activity.


   Acknowledgments
 
We would like to thank Terrance Phernetton and Dr. Ronald Magness for assistance in animal preparation.


   Footnotes
 
This work was supported by Grants USDA 0002159 (to I.M.B.), National Institutes of Health (NIH) HL64601 (to I.M.B.), NIH HD 38843 (to I.M.B.), and American Heart Association predoctoral fellowship 0010154Z (to J.A.S.). This paper forms part of the studies of J.S. toward a Ph.D. in the Endocrinology Reproductive Physiology Training Program.

All authors have nothing to declare with regard to this manuscript.

First Published Online February 2, 2006

Abbreviations: Ad, Adenovirus; AII, angiotensin II; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester); bFGF, basic fibroblast growth factor; ca-Akt, constitutively active Akt; EGF, epidermal growth factor; eNOS, endothelial NOS; GFP, green fluorescent protein; IP3, inositol triphosphate; IP3-R, IP3 receptor; MEK, MAPK kinase; NO, nitric oxide; NOS, NO synthase; NOx, total nitrate and nitrite converted to a reduced species; NP-UAEC, nonpregnant UAEC; PI3-K, phosphatidylinositol 3-kinase; PLC, phospholipase C; P-UAEC, pregnant UAEC; UAEC, uterine artery endothelial cells; VEGF, vascular endothelial growth factor.

Received April 6, 2005.

Accepted for publication January 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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