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Endocrinology Vol. 147, No. 11 5228-5235
Copyright © 2006 by The Endocrine Society

Inverse Relationship between Nitric Oxide Synthases and Endothelin-1 Synthesis in Bovine Corpus Luteum: Interactions at the Level of Luteal Endothelial Cell

Maya Rosiansky-Sultan, Eyal Klipper, Katharina Spanel-Borowski and Rina Meidan

Department of Animal Sciences (M.R.-S., E.K., R.M.), Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel; and Institute of Anatomy (K.S.-B.), University of Leipzig, D-04103 Leipzig, Germany

Address all correspondence and requests for reprints to: Rina Meidan, Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel. E-mail: rina.meidan{at}huji.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endothelin-1 (ET-1) and nitric oxide (NO) play pivotal roles in corpus luteum (CL) function. The present study examined the interplay between NO and ET-1 synthesis in the bovine CL. We found similar inducible and endothelial NO synthase (iNOS and eNOS, respectively) activities in the young CL (d 1–5) expressing the highest levels of both eNOS and iNOS mRNA. These values later declined at mid-cycle (d 8–15) and remained low at later stages (d 16–18). Luteolysis, initiated by prostaglandin F2{alpha} analog administration, further reduced NOS mRNA and by 24 h, NOS values dropped to approximately 15% of those at mid-cycle. eNOS protein levels followed a similar pattern to its mRNA. Because endothelial cells (ECs) are the main site for ET-1 and NO production in the CL, we examined the direct effects of the NO donor, NONOate on luteal ECs (LECs). Elevated NO levels markedly decreased ET-1 mRNA, and peptide concentrations in cultured and freshly isolated LECs in a dose-dependent manner. In agreement, NOS inhibitor, NG-nitro-L-arginine methyl ester, stimulated ET-1 mRNA expression in these cells. Interestingly, NO also up-regulated prostaglandin F2{alpha} receptors in LECs. These data show that there is an inverse relationship between NOS and ET-1 throughout the CL life span, and imply that this pattern may be the result of their interaction within the resident LECs. NOS are expressed in a physiologically relevant manner: elevated NO at an early luteal stage is likely to play an important role in angiogenesis, whereas reduced levels of NO during luteal regression may facilitate the sustained up-regulation of ET-1 levels during luteolysis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CORPUS LUTEUM (CL) growth is accompanied by extensive angiogenesis, expressed in the dense network of capillaries surrounding the steroidogenic cells (1, 2, 3). More than merely constituting luteal blood vessels, these endothelial cells (ECs) have unique properties that enable them to play key roles in the function of this endocrine gland (4, 5, 6, 7).

Endothelin-1 (ET-1) and nitric oxide (NO) are two endothelium-derived mediators that act as mutual antagonists in maintaining vascular tone and are involved in numerous other physiological and pathological processes (8, 9, 10). In the CL, these vasoactive compounds modulate progesterone production and were suggested as mediators in the process of luteolysis (11, 12, 13, 14, 15). Most of the ET-1 and NO present in luteal tissues are produced locally by the resident ECs (5). Interestingly, we have recently shown that the ability of these cells to synthesize both ET-1 and NO is affected by the surrounding luteal microenvironment (5).

ET-1 is a pleiotropic peptide, best known for its vasoconstrictor activity; however, it has other diverse biological functions including embryonic development, cardiovascular homeostasis and vascular permeability (16, 17, 18). ET-1 mediates its various effects via two G protein-coupled receptors: ETA and ETB (19, 20). The mature ET-1 peptide is synthesized from an approximately 200-amino acid pre pro ET-1 (ppET-1) that is proteolytically cleaved into the inactive 38-amino acid big ET-1, subsequently cleaved at the Trp-Val by a zinc-binding metalloendopeptidase, ET-converting enzyme-1 (ECE-1), to form the 21-amino acid mature, active peptide (21, 22). The ECE-1 null mice exhibit a phenotype similar to that of ET-1 or ETA-deficient mice, thus demonstrating the physiological relevance of ECE-1 in generating bioavailable ET-1 (18).

Like ET-1, NO regulates multiple biological functions. This gaseous molecule is a potent vasodilator, plays a critical role in angiogenesis, and mediates cell growth and apoptosis (23, 24, 25). The formation of NO is controlled at the level of NO synthase (NOS) (26, 27). Two NOS isoforms were identified in the CL: the constitutive, calcium (Ca2+)-dependent endothelial NO synthase (eNOS, type III NOS), synthesizes intermittent low levels of NO. In contrast, the inducible NOS (iNOS, type II NOS) is formed after activation by proinflammatory cytokines such as interferon {gamma}, IL-1, or TNF{alpha}, in many cell types including ECs (26, 27, 28). Once expressed, iNOS can generate locally high concentrations of NO for extended periods of time (23). In vitro, iNOS-expressing cells can produce up to 5 µM of steady-state NO concentrations (29).

Many studies employing several species demonstrated a consistent pattern of luteal ET-1 expression, with rapid up-regulation during luteolysis, either natural or prostaglandin F2{alpha} (PGF2{alpha})-induced, as the prominent feature (11, 15, 30, 31, 32, 33). In contrast, reports of NO production and NOS expression throughout the cycle are controversial (34, 35, 36, 37).

This present study examined the relationship between NO and ET-1 synthesis in the bovine CL. As a first step, the levels of eNOS and iNOS expression throughout the bovine cycle and after PGF2{alpha}-induced luteolysis were quantified. To better understand the profiles of these EC-derived factors in the CL, we also examined the relationship between NO and ET-1 using two cells models: 1) ECs freshly isolated from the CL; and 2) ECs maintained in long-term culture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
CLs were collected from Holstein dairy cows at a local slaughterhouse and the luteal stage was determined by macroscopic examination (38). The following groups were examined: early-lutal phase (d 1–5), mid-luteal phase (d 8–15), and late-luteal phase (d 16–18). To induce luteolysis, Holstein dairy cows exhibiting regular cycles were used. Their estrous cycles were synchronized by progesterone and Cloprostenol-Estrumate (Coopers, Berkhamsted, UK), a PGF2{alpha} analog (PGF2{alpha}-A), as previously described (30). Cows at the mid-luteal phase (d 8–15) were injected with PGF2{alpha}-A and CL were collected 4 h (n = 5), 24 h (n = 3), and 48 h (n = 2) later. CL samples were snap-frozen in liquid nitrogen and stored at –80 C until RNA extraction. All animal studies described here were reviewed and approved by the appropriate Institutional Animal Care and Use Committee.

Enrichment of LECs
For enrichment of luteal cell subpopulations, mid-cycle CLs were dispersed by means of collagenase IV (Worthington Biochemical Corp., Freehold, NJ) as previously described (39). Briefly, CLs were sliced and dispersed in medium M-199 (Life Technologies, Inc., Gaithersburg, MD) containing 0.5% BSA (Sigma, St. Louis, MO) and collagenase IV (420 U/ml). Dispersed luteal cells were mixed with epoxy magnetic beads (Dynabeads M-450; Dynal, Oslo, Norway) that had been precoated with Bandeiraea simplicifolia lectin-1 (BS-1; Vector Laboratories, Burlingame, CA). A fraction of BS-1-positive cells (enriched ECs) was collected for RNA extraction and the rest were cultured. Cells designated for culture were seeded onto plates precoated with 2% Vitrogen type I collagen (Cohesion Technologies, Palo Alto, CA) and cultured overnight in endothelial-serum-free medium basal growth medium (EBM; Invitrogen Corp., Paisley, UK) containing 2.5% fetal bovine serum (FBS) and 1 µl/ml supplements (Cambrex Corp., East Rutherford, NJ). ECs were cultured overnight in media containing a nitric oxide donor, spermine NONOate (NONOate; Cayman, Ann Arbor, MI). At the end of the incubation period, RNA was extracted from the cells and the culture medium was collected to determine the ET-1 peptide concentration.

Long-term endothelial cell cultures
Endothelial cells derived from bovine CL were obtained as previously described (7, 40). These cells maintain a stable phenotype during long-term culture. Cells were grown on plates precoated with 2% Vitrogen in DMEM-F12 containing 10% FBS and 2 mM glutamine (Biological Industries, Beit Haemek, Israel). Experiments were carried out on cells from passages five to 12. First, cells were plated onto six-well plates and grown to 70–80% confluence, then washed and preincubated overnight with DMEM-F12 containing 0.1% FBS and 0.5% BSA. Next, the medium was replaced with fresh medium containing varying concentrations of NONOate or NOS inhibitor [NG-nitro-L-arginine methyl ester (L-NAME)], and the cells were cultured as indicated. Finally, the culture medium was collected to determine the ET-1 peptide concentration, and RNA was extracted from the cells.

RNA isolation and real-time PCR
Total RNA was isolated from the cells with TriReagent (MRC, Cincinnati, OH) according to the manufacturer’s instructions. PCRs were performed using the PE Biosystems’ GeneAmp 5700 sequence detection system, as described by Klipper et al. (5). Briefly, each real-time reaction (18 µl) contained SYBR Green Master Mix (Eurogentec, Seraing, Belgium) (200 µM deoxynucleotide triphosphates, 5 mM MgCl2, uracil N-glycosylase, and Amplitaq HotGoldStar DNA polymerase) to which 0.54 µl of a 1:10,000 dilution of SYBR Green stock solution, 1.5 mM deoxynucleotide triphosphates, 10 nM of each primers, and 25–50 ng cDNA were added. Glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene was used as the standard. Gene expression in enriched LECs was normalized to CD31, an endothelial cell marker, to normalize for endothelial gene expression and not to any steroidogeic cells cDNA contamination. Dissociation curve analysis was performed after each real-time experiment to confirm the presence of only one product and the absence of primer dimmers. The threshold cycle number (Ct) for each tested gene X was used to quantify the relative abundance of the gene: 2–(Ct gene X-CtG3PDH) *1000. Table 1Go presents the list of primers.


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  		TABLE 1. Primer list for real-time PCR

 
Determination of the ET-1 peptide concentration
Determination of the ET-1 peptide concentration in the cells’ culture medium was carried out by the Endothelin EIA kit (Cayman, Ann Arbor, MI) according to the manufacturer’s instructions. The specificity of the antibody to endothelin-1, -2, and -3 is 100% and less than 0.01% for big endothelin. Endothelial cells produce almost exclusively ET-1; therefore, it is likely that endothelin-2,3 levels were negligible.

Western blot analysis
Pieces of the tissues at different stages of the cycle as follows: early (n = 3), mid (n = 3), late (n = 3), and regressed CL (n = 4) were homogenized (20 mg/ml) in lysis buffer (20 mM Tris-HCl (pH 8.6), 0.5% sodium dodecyl sulfate, 1:100 protease inhibitor cocktail (Sigma) and 1:100 phosphatase inhibitor cocktail II (Sigma). The homogenates then centrifuged for 10 min at 2000 x g (4 C) and protein concentrations in the supernatants were determined by Bio-Rad (Hercules, CA) DC reagents. All steps were preformed on ice, and samples were kept in –80 C until use. Fifty micrograms of protein of each sample were separated by 7.5% SDS-PAGE, under reducing conditions. Thereafter, the samples were electrically transferred to nitrocellulose membranes. The membranes were cut at the 72-kDa marker to obtain upper and lower parts. After 1 h of blocking in TBST + 5% low-fat milk, the upper parts of the membranes were incubated overnight on a rocking platform, with anti-eNOS antibody (1:1000; Cell Signaling, Boston, MA). The lower part of the membranes were incubated with an antibody to p42/44-total MAPK (1:70,000; Sigma). Blots were incubated thereafter for 2 h (24 C) with horseradish peroxidase-conjugated goat antirabbit IgG. A chemiluminescent signal was generated with EZ-ECL (Biological Industries) and then the membranes were exposed to x-ray film.

NOS activity assay
NOS activity was assessed by measuring the conversion of [14C] L-arginine to [14C] L-citrulline, according to Giraldez and Zweier (41) with slight modifications. CL of early phase were collected at a local slaughterhouse and snap frozen in liquid nitrogen. The tissues were ground while still frozen, and then homogenized in 1ml ice cold homogenization buffer: 50 mM Tris (pH 7.4), 0.1 mM EDTA, 0.1 mM EGTA, and protease inhibitor (1:50). Three hundred micrograms of tissue homogenate in 100 µl of homogenization buffer were put in a test tube and the final volume of 300 µl was obtained by adding: 50 mM Tris (pH 7.4), 3 mM reduced nicotinamide adenine dinucleotide phosphate (Sigma), 200 µM CaCl2, 30 µM EDTA, 30 µM EGTA, 100 nM calmodulin (Sigma), 4 µM flavin adenine dinucleotide (Sigma), 4 µM flavin adenine mononucleotide (Sigma), 3 µM tetrahydrobiopterin (Sigma), and 0.1 µCi [14C]L-arginine(Amersham). After 50 min of incubation at 37 C, the reaction was stopped by adding 2 ml ice-cold stop buffer [20 mM HEPES and 2 mM EDTA (pH 5.5)]. Subsequently the samples were passed through a column of the cation-exchange resin Dowex AG 50WX-8 (Bio-Rad). The column was then washed with additional 2 ml ddH2O (double-distilled water). The radioactivity corresponding to [14C] L-citruline content in the solution collected from the column was measured after adding 10 ml scintillation liquid [Ultima Gold; Packard (by PerkinElmer, Wellesley, MA)]. [14C] L-citruline production was also tested with 5 mM EGTA to determine the Ca2+-independent (iNOS) activity. Calcium-dependent (eNOS) activity was calculated as the difference between that measured with CaCl2/Calmodulin and that measured in the EGTA buffer, whereas Ca2+-independent (iNOS) activity was calculated as the difference between that measured in the EGTA buffer and that measured with 250 µM L-NAME (pan NOS inhibitor).

Statistical analyses
Data are presented as means ± SEM. The one-way ANOVA Tukey-Kramer test was used to determine the statistical difference between treatments, as indicated in Results and the legends to the figures. Additionally, the Student’s t test was used to evaluate the diverse expression of genes between dispersed LECs and overnight cultured LECs. The differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Profiles of iNOS and eNOS in CL throughout the bovine luteal phase and after PGF2{alpha}-A administration
The levels of mRNA for eNOS and iNOS in CL collected at early, mid-, and late luteal phases were determined by real-time PCR using specific primers (Fig. 1AGo). The levels of eNOS were higher (10-fold on the average) than those of iNOS at each of the time points examined. The expression of mRNA for both NOS enzymes declined progressively as the CL aged, but at different rates. At the mid-luteal phase, the eNOS levels were reduced by approximately 70% compared with the early stage and remained stable thereafter. On the other hand, iNOS only slightly declined at mid-phase, and a marked 6-fold reduction, compared with the early stage, was observed in late luteal phase (Fig. 1AGo).


Figure 1
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  		FIG. 1. Expression of iNOS and eNOS mRNA throughout the lifespan of CL (A) and PGF2{alpha}-A induced luteolysis (B). A, iNOS (left y-axis) and eNOS (right y-axis) mRNA levels were determined in CL during the early (d 1–5, n = 9), mid- (d 8–15, n = 10), and late (d 16–18, n = 5) luteal phases. B, iNOS and eNOS mRNA levels, 4 h (n = 5), 24 h (n = 3), and 48 h (n = 2) after administration of PGF2{alpha}-A, and displayed as the percent of gene expression in the mid-luteal phase CL (nontreated cows). mRNA levels were determined by real-time PCR. The results represent the mean ± SEM. Means with no common letters (a and b for iNOS and italic, a–c for eNOS) differ significantly between the luteal phases (A) or times after PGF2{alpha}-A (B).

 
Luteolysis induced by injection of PGF2{alpha}-A further reduced the expression of iNOS and eNOS and 24 h later, their levels were lowered to 20% and 10%, respectively, of those observed in nontreated cows (Fig. 1BGo). This effect was not immediate because 4 h after administration of PGF2{alpha}-A NOS mRNAs were still not affected (Fig. 1BGo). Luteolysis was confirmed by declining plasma progesterone concentrations and luteal steroidogenic acute regulatory protein mRNA (data not shown).

The ratio of eNOS (Ca2+ dependent) and iNOS (Ca2+ independent) in total luteal NOS activity was then measured, as demonstrated in Fig. 2Go, both enzymes contributed almost equally to NOS activity in the young CL. In regressed CL, NOS activity was too low to be accurately divided into e/iNOS (data not shown).


Figure 2
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  		FIG. 2. Relative iNOS and eNOS activity in CL at early luteal phase. NOS activity was determined by the conversion of [14C] L-arginine to [14C] L-citrulline. Calcium-dependent (eNOS) activity was calculated as the difference between the activity measured with CaCl2/Calmodulin and that measured in the EGTA buffer, whereas Ca2+-independent (iNOS) activity was calculated as the difference between that measure in the EGTA buffer and that measure with 250 µM L-NAME (total NOS inhibitor). Results are the mean ± SEM from three different CL assayed independently and displayed as percent of total NOS activity.

 
eNOS protein levels followed a similar pattern to its mRNA shown in Fig. 1Go but with a lag of time, high in early and mid-luteal phases, lower in the late stage, and lowest in regressed CL (Fig. 3Go). Protein samples from these time points were also probed with anti phospho-ser-1177-eNOS but were all found to be negative. In contrast, phospho eNOS was readily detected in LECs incubated with VEGF (data not shown).


Figure 3
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  		FIG. 3. Expression of eNOS protein levels during different stages of the luteal phase. Upper panel, A representative Western blot; 50 µg of protein from each sample were loaded per well on 7.5% gels. The gels were electroblotted, and Western analyses were performed with anti bovine eNOS antibody. Lower panel, Quantification of eNOS protein. Results were then scanned and densitometric analyses were performed with NIH Image software (National Institutes of Health, Bethesda, MD). Differences in loading were normalized to MAPK using total MAPK antibodies. Results represent means ± SEM for three different CL at each luteal phase. Means with no common letters (a and b) differ significantly.

 
Effects of NONOate on ET-1 and prostaglandin F receptor (FP) expression by LECs
We next sought to determine the levels of the two NOS enzymes along with those of ppET-1 in LECs, obtained from mid-cycle CLs. The levels of ppET-1 were low in luteal ECs freshly isolated from the CL, but were greatly elevated during overnight culture (Fig. 4Go). The expression of mRNA for NOS followed an opposite trend: the levels of both iNOS and eNOS mRNA were high in freshly isolated cells but were markedly reduced during culture (Fig. 4Go). The relationship between iNOS and eNOS in LECs was as in the CL, with eNOS mRNA levels approximately 10-fold higher than those of iNOS (Figs. 1Go and 4Go).


Figure 4
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  		FIG. 4. A comparison of ppET-1 and NOS mRNA expression in freshly isolated and overnight cultured LECs. mRNA levels for ppET-1 (upper panel), iNOS and eNOS (lower panel) were determined by real-time PCR and normalized to CD31. The results represent the mean ± SEM from three separate experiments. Asterisks denote significant differences (*, P < 0.005; **, P < 0.001).

 
To examine whether elevated NOS levels in LECs might have suppressed the expression of ET-1, we manipulated intracellular NO levels using NO donor (spermineNONOate) and a NOS inhibitor (L-NAME). Freshly isolated LECs were treated for 24 h with varying concentrations of the NO donor. The data indicated a dose-dependent inhibition of ppET-1, with more than 90% inhibition observed at 100 µM (Fig 5Go). In agreement with the mRNA data, increasing NONOate concentrations also significantly reduced the levels of the ET-1 peptide released into the culture medium (Fig. 5Go).


Figure 5
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  		FIG. 5. Effect of NONOate (NO donor) on ET-1 expression in freshly isolated luteal ECs. Freshly isolated luteal ECs were grown overnight in basal endothelial growth medium (EBM; 2.5% FBS, 1 µl/ml supplements) containing NONOate (0–100 µM). The results represent the mean ± SEM from three separate experiments and are displayed as percent of control. Different letters indicate significant differences between treatments (a–c for ppET-1 mRNA and italics for ET-1 peptide).

 
Next, we examined the effects of NONOate on the LECs lines maintained in long-term cultures. Elevating cellular NO levels by NONOate in these cells reduced both the ppET-1 and ECE-1 mRNA levels in a dose-dependent manner (Fig. 6AGo). ET-1 peptide levels were concomitantly reduced in these cells (Fig. 6BGo). These findings corroborate those observed in freshly isolated cells (Fig. 5Go), although at a lower magnitude. For instance, in long-term cultured LECs, 300 µM of NONOate were required to achieve a 50% inhibition in ET-1 peptide, compared with 80% with 100 µM in freshly isolated cells. This might be due to an elevation in the ppET-1 levels concurrent with an adaptation of the cells to culture.


Figure 6
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  		FIG. 6. Effect of NONOate (NO donor) on ET-1 expression in long-term cultured luteal ECs. Cells were serum-starved overnight and then incubated in basal media (DMEM-F12, 0.1% FBS, 0.5% BSA) containing NONOate (0–300 µM) for 24 h. A, Levels of mRNA for ppET-1 and ECE-1. B. ET-1 peptide concentrations in culture media. Data (mean ± SEM; n = 4) are displayed as the percent of control (0 µM NONOate). Means with no common letters (a–c) differ significantly between treatments.

 
Incubation of LECs with the NOS inhibitor, L-NAME, elevated ppET-1 mRNA levels after 4 and 8 h of incubation, with a maximal 1.6-fold stimulation induced by 3 mM of the compound (Fig. 7Go). Again the modest elevation may be due to the elevated basal ppET-1 levels.


Figure 7
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  		FIG. 7. Effect of L-NAME (NOS inhibitor) on ppET-1 mRNA levels in long-term cultured luteal ECs. Cells were serum-starved overnight and then incubated in basal media (DMEM-F12, 0.1% FBS, 0.5% BSA) containing 0–3 mM L-NAME for 4 and 8 h. Data (mean ± SEM; n = 3) are displayed as the percent of control. Means with no common letters (a–c) differ significantly between treatments.

 
Incubating LECs with NONOate did not affect NOS expression (data not shown); however, the levels of mRNA for PGF2{alpha} receptor (FP receptor) in these cells rose in a dose-dependent manner up to 300 µM NONOate, at which point the FP receptor mRNA increased 4-fold (Fig. 8Go).


Figure 8
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  		FIG. 8. Effect of NONOate (NO donor) on the FP receptor mRNA levels in luteal ECs. Long-term cultured luteal ECs were serum starved overnight and then cultured in basal media (DMEM-F12, 0.1% FBS, 0.5% BSA) containing NONOate (0–300 µM) for 24 h. The results are displayed as the percent of control and represent the mean ± SEM from four separate experiments. Means with no common letters (a–c) differ significantly between the different treatments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study depicts the profiles of NOS expression in the bovine CL during the estrous cycle, indicating that both eNOS (at the mRNA and protein levels) and iNOS mRNA were high in the early luteal stage and were progressively reduced later in the cycle. Induction of luteolysis by administering PGF2{alpha}-A further reduced the levels of NOS expression.

This study is the first to determine both luteal eNOS and iNOS mRNA levels at different stages of the estrous cycle and after induced luteolysis, using quantitative real-time PCR. Our data are in a good agreement with several other studies that examined certain phases of the cycle. Johnson et al. (42) reported that the expression of eNOS mRNA in sheep CL was greater on d 2–4 than on d 10 and 15 of the estrous cycle. Neuvians et al. (36), in examining the iNOS levels in bovine CL after PGF2{alpha}-A-induced luteolysis, found a gradual reduction from 2–64 h after injection. In the regressed rabbit CL, eNOS mRNA levels were similarly reduced (34). However, in contrast to the mRNA levels, the data available in the literature on the protein expression of NOS enzymes (determined by either Western blotting or immunostaining) were conflicting and not always consistent with the mRNA data (34, 37, 43). This may be due to a lack of species-specific antibodies. A meticulous study (44) comparing 10 different anti-NOS antibodies found unexpected immunoreactivities in terms of both cell targets and molecular mass when the species examined differed from the species the antibodies were originally raised against. Indeed, using bovine-specific anti-eNOS antibodies, there was a good agreement between eNOS mRNA and protein levels. NOS activity in the CL measured here demonstrates that eNOS and iNOS contributed equally to total NOS activity in young CL despite the fact that eNOS mRNA in this gland was significantly higher than iNOS. This can be anticipated because iNOS is much more robust enzyme than eNOS (23, 29).

The addition of NO donors caused a dose-dependent decrease in both estradiol and progesterone secretion in cultured human granulosa-luteal cells (45). A similar inhibitory effect by the NO donor on the secretion of progesterone was also observed in luteinized rat granulosa cells (46) and in mixed cells of bovine CL (47). Moreover, perfusion of bovine CL with a NOS inhibitor (L-NAME) inhibited PGF2{alpha}-induced luteolysis, stimulated progesterone, and extended the estrous cycle when used during the late luteal phase (37, 48). Based on these studies, it was postulated that NO is involved in luteolysis. The profile of NOS depicted here, i.e. iNOS and eNOS being high at the early luteal phase and low during luteal regression does not support this contention. However, this does not rule out a transient increase in NO that can be achieved, for instance, via phosphorylation of the enzyme. A transient increase in luteal blood flow, observed at 1–4 h after PGF2{alpha} injection (49), was attributed to the vasodilatory effect of NO. In regressed CL examined in this study (24–48 h after PGF2{alpha}-A injection), we could not detect phospho-ser-1177-eNOS. However, whether such an increase has occurred at earlier time points is still unknown. One should keep in mind that, in contrast to the possible transient up-regulation of NO during luteolysis, ET-1 is rapidly elevated [2–4 h after PGF2{alpha} (50, 51)] and is maintained at high levels for more than 3 d. Increased NO production during the early stages of the cycle, suggested by the elevated NOS levels, is likely to play an important role in the different stages of angiogenesis by modulating vascular hyperpermeability, migration, proliferation, and the organization of ECs into a network structure (52, 53, 54). It was also suggested that NO plays a critical role in vascular endothelial growth factor-induced angiogenesis (55). The data reported here also showed that elevated NO levels up-regulated FP receptors in cultured ECs. Luteal FP receptors are induced soon after the LH surge, mainly as a result of LH/cAMP action on steroidogenic cells (56, 57, 58). Data presented here suggest that induction of these receptors in LECs by NO can contribute to the increase of FP receptors in the developing CL. In contrast to PGF2{alpha} of uterine origin, luteal PGF2{alpha} was shown to have luteotropic effects (59, 60, 61, 62). One of these could be an increase in the proliferation of LECs, as was recently suggested (4).

Comparing the schematic pattern of the NOS levels, reported in this study, together with the pattern of the ET-1 levels, we and others have reported previously (11, 15, 51, 63) Fig. 9Go) suggests an inverse relationship between these two compounds. This pattern also suggests that ET-1 and NO may modulate the production of each other. Both are produced within the same cell type (ECs), which supports this proposition. An inverse relationship between ppET-1 and NOS was indeed observed in freshly isolated LECs and in cultured LECs (5 and this study). The present study extends these observations by showing that elevating the NO levels in LECs with spermine NONOate decreased ET-1 synthesis at the level of its gene expression, and of its converting enzyme, which ultimately decreased peptide secretion. The inhibitory effect of elevated NO levels was very pronounced in cells isolated from the CL but was also significant in cells maintained in culture for longer time. These data are consistent with the stimulatory effects on ppET-1 induced by the NOS inhibitor, L-NAME, which we have observed in LECs and also with other reports in different systems indicating that NO limits the net production of ET-1 (64, 65). The underlying mechanism responsible for this phenomenon may involve activation of soluble guanylate cyclase and increased cGMP generation by NO, as demonstrated before in ECs exposed to exogenous NO (66). In fact, it is physiologically plausible because ET-1 and NO act as mutual antagonists in determining many processes including vascular tone (8, 67), atherosclerosis, platelet activity, as well as leukocyte chemotaxis (68, 69).


Figure 9
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  		FIG. 9. Schematic representation of the relative levels of eNOS mRNA and protein, iNOS mRNA and ET-1 peptide throughout the luteal life span.

 
To date, the possibility of NO inhibiting ET-1 synthesis in the CL has not been directly examined. The in vivo data suggest that this may indeed be the case because high NOS levels in the early luteal stage might account for the low ET-1 levels that characterize this phase (63, 70). Conversely, reduced levels of NOS during luteal regression may facilitate the elevation of ET-1 levels, a hallmarks of luteolysis (13, 70). The inhibitory effects of ET-1 on luteal progesterone biosynthesis both in vitro and in vivo are well established (13, 71). Therefore, the low amounts of ET-1 peptide present in the young CL and the absence of its induction by PGF2{alpha} were suggested to confer, at least partially, resistance to luteolytic signals.

The inverse relationship between NOS and ET-1 throughout the CL life span may result from the inhibition exerted by NO on ET-1 synthesis in the resident LECs. Therefore, a better understanding of the balance between NO and that of ET-1 in the ECs may have implications for luteal physiology.


   Footnotes
 
This study was supported by Grant 0376235 from the Bi-National Agricultural Research & Development Foundation (BARD).

Disclosure summary: all authors have nothing to declare.

First Published Online August 3, 2006

Abbreviations: CL, Corpus luteum; ECs, endothelial cells; ECE-1, ET-converting enzyme-1; eNOS, endothelial NOS; ET-1, endothelin-1; FBS, fetal bovine serum; FP, prostaglandin F receptor; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; iNOS, inducible NOS; LECs, luteal ECs; L-NAME, NG-nitro-L-arginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; PGF2{alpha}, prostaglandin F2{alpha}; ppET-1, pre pro ET-1.

Received June 14, 2006.

Accepted for publication July 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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