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a Departments of Obstetrics and Gynecology and
b Physiology and Pharmacology, Perinatal Research Laboratory, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All three isoforms of NOS have been identified alone or in combination in the uterus of many species [1–6]. In general, at least one NOS isoform has been found in endometrial stroma, endometrial glands, decidua, decidual macrophages, vascular endothelial cells, and myometrial smooth muscle cells. The factors controlling the expression of the different NOS isoforms and the physiological role of the NO produced in the different uterine compartments are not clearly established. Species differences are particularly important regarding type II NOS expression. Sheep and goats are distinguished among ruminants by the weak iNOS response observed in activated macrophages in vivo and in vitro [7, 8].
We have previously shown that estrogen replacement increases NOS activity in myometrium, but not endometrium, of nonpregnant ovariectomized sheep, suggesting that NOS expression within the uterus is under different regulatory mechanisms [9]. In the present study, we sought to determine 1) which NOS isoform(s) increases in response to estrogen, 2) if the increase in NOS protein is associated with an increase in steady-state mRNA levels for that particular isoform, and 3) if the effects of estrogen are isoform and uterine compartment-specific, namely, myometrium, glandular endometrium, and caruncles (nonglandular endometrium).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All the procedures for housing and handling of animals, surgical implantation of catheters, and postoperative management were approved by Wake Forest University's Institutional Animal Care and Use Committee. Twenty nulliparous nonpregnant sheep with an average weight of 50 kg were synchronized in their estrous cycles with 6-mg norgestomet implants (Syncromate; Ceva Laboratories Inc., Overland Park, KS). The implants were removed 28–30 days before surgery to allow the occurrence of one spontaneous estrus 2 wk before surgery. Animals were brought into the laboratory and placed in metabolism cages with free access to food and water. Twenty-four hours before surgery, feed and water were withheld. Sheep were premedicated with 2.5 mg of atropine and 1 g of ketamine, and operated on under halothane general anesthesia (1.5–2% in 2 L/min of oxygen). The presence of a mature or a recently ruptured follicle was confirmed, and bilateral oophorectomy was performed. In addition, catheters were placed 25 cm into the femoral vein and femoral artery. Starting on Day 4 after surgery, sheep were treated i.v. with 100 µg/24 h estradiol-17ß in NaCl 0.9% containing 0.025% ethyl alcohol for either 5 or 8 days (n = 6 each estrogen treatment group), or with vehicle (n = 8) at a rate of 1 ml/h as previously described [10]. At the end of the treatment, sheep were anesthetized with ketamine and halothane, and the genital tract was removed. After the main uterine artery and vein and their branches were removed, the uterus was dissected into myometrium, and caruncular and noncaruncular endometrium and immediately frozen in liquid nitrogen and stored at -80°C.
Ribonuclease Protection Assay (RPA)
In vitro transcription was carried out using Ambion's maxiscript protocol. Briefly, the mixture contained 2 µl 10-strength transcription buffer, 0.5 mM each of the nucleotides ATP, CTP, and GTP, 2.5 µM cold UTP, 0.5 µg linearized sheep type I NOS or type III NOS (Dr. Krimpamoy Aguan, University of Maryland at Baltimore), 7 µl 3000 Ci/mmol [32P]UTP (Dupont, NEN, Wilmington, DE), and 2 U T7 or T3 RNA polymerase in a total volume of 20 µl. The reaction was carried out at 37°C for 1 h; then 1 U DNase I was added and incubated for 15 min at 37°C. Unincorporated [32P]UTP was removed by column chromatography (Spin Columns Boehringer, Indianapolis, IN), and the probe was further purified using 5% PAGE/8 M urea gel. Tissue (75 mg) was homogenized in 1 ml TRIzol reagent (Sigma, St. Louis, MO) and extracted with chloroform, and the aqueous phase was mixed with isopropyl alcohol. The RNA pellet was washed with 75% ethanol, air-dried, and resuspended in ribonuclease (RNase)-free water. PolyA RNA was extracted from 400 µg total RNA using 100 mg oligo(dT) cellulose (MicroPoly(A)Pure; Ambion Inc., Austin, TX). Hybridization was performed by mixing 5 µg polyA RNA and 2 x 105 cpm type I NOS or type III NOS probe in 20 µl hybridization buffer, heating at 95°C for 4 min, and incubating overnight at 42°C. Samples were then digested with diluted RNase A/T mixture and centrifuged with 10 µg of glycogen at 12 000 x g. Pellets were air-dried, resuspended in 8 µl of formamide-based loading buffer, and loaded onto 5% acrylamide/8 M urea denaturing gels. In all gels, 2000 cpm of a molecular weight marker ([32P]UTP-labeled Ambion's Century Marker) was included, and gels were exposed to film at -70°C with an intensifying screen. All bands with intensities within the linear range were scanned to determine optical density.
Western Immunoblotting
Western blot analysis was performed according to the method of Laemmli [11] using 8.0% SDS-PAGE. Tissue samples were homogenized in Tris buffer (50 mM Tris-hydrochloric acid, 0.1 mM EDTA, 0.1 mM ethylene glycol-bis [ß-aminoethyl ether] N,N,N',N'-tetraacetic acid [EGTA], 0.5 mM dithiothreitol [DTT], 150 mM NaCl, 1% Triton X, 0.1% SDS, 1% sodium deoxycholate, 12 mM 2-mercaptoethanol, 2 µM leupeptin, 1 µM pepstatin, and 1 mM PMSF). Samples (400–600 mg) were placed in liquid nitrogen and crushed in a stainless steel mortar, and the powder was homogenized in 2–3 ml of buffer with a Tissue Tearor (Biospec Products, Inc., Bartlesville, OK). The homogenate was centrifuged at 2000 x g to remove cellular debris and then at 15 000 x g for 20 min. The supernatant was collected, and protein concentration measured with the bicinchoninic acid method using BSA as standard (Pierce, Rockford, IL). Protein aliquots were mixed 1:4 in loading buffer, separated in 8% Tricine gels (Novex, San Diego, CA), and blotted onto polyvinylidene fluoride (PVDF) membranes (Immobilon; Millipore; Marlborough, MA) by semidry electroblotting. Blots were blocked overnight at 4°C with 6% dry nonfat milk, rinsed with Tris-buffered saline/0.05% Tween 20, and incubated for 2 h at room temperature with primary antibody (Transduction Laboratories, Lexington, KY; type I NOS 1:2000 and type III NOS 1:750) and for 1 h with horseradish peroxidase-conjugated second antibody. A positive reaction was identified with enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL), and relative intensity was normalized by loading equal protein amounts and by the intensity of the standard preparation for that isoform (fetal cerebellum or purified bovine brain NOS [Sigma] and a lysate of bovine endothelial cells) in each gel. All bands with intensities within the linear range were scanned to determine optical density.
Statistical Analysis
Data represent optical density and are expressed as mean ± SEM. All data were analyzed using one-way ANOVA, and treatments were compared to control using Tukey's test. Differences were considered statistically significant if the change in optical density was greater than 30% and p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western blot analysis demonstrated that type I NOS and type III NOS are the predominant isoforms in the uterus of the nonpregnant sheep. The anti-type I NOS antibody recognized a band of approximately 160 kDa in myometrium, endometrium, and caruncular endometrium; however, very often a secondary band of 130–150 kDa was observed. This secondary band was present in those tissues in which type I NOS was more abundant, in the commercially available type I NOS, and in the fetal cerebellum used as loading control. However, this band was not present in rat recombinant type I NOS (Stratagene, La Jolla, CA; data not shown). Whether this band represents a degradation product, a post-translational modification, or a splice variant is presently unknown. In all uterine compartments, type I NOS was found in the 100 000 x g supernatant (soluble in cytoplasm) and in the 100 000 x g pellet (cell membrane) in relatively equal proportions (data not shown). The anti-type III NOS antibody recognized in all uterine compartments a single protein band of approximately 135 kDa, as in the endothelial cell lysate used as loading control and predominantly associated with the 100 000 x g pellet (data not shown). No specific protein band was observed using three different antibodies raised against human or murine iNOS (monoclonal and polyclonal from Transduction Laboratories, Lexington, KY; polyclonal from Oxford Scientific, Oxford, MI; data not shown). In the RPA, the type I NOS-protected fragment had the same size in uterine tissues as it did in fetal cerebellum, and the type III NOS-protected fragment had the same size in sheep aortic endothelium as it did in all uterine tissues.
Myometrium
Administration of estradiol-17ß for either 5 or 8 days significantly increased steady-state mRNA levels of type I NOS (Fig. 2, left panel). Using Western immunoblotting, a coordinated increase (p < 0.05) in type I NOS protein mass was also observed (Fig. 2, right panel). Type I NOS steady-state mRNA levels and protein mass increased to approximately 3-fold in the 5-day replacement group and to 4-fold in the 8-day replacement group (p < 0.05 by one-way ANOVA and Tukey's test). In contrast, mRNA and protein mass for type III NOS showed a progressive increase that reached statistical significance in the 8-day replacement group (p < 0.05; Fig. 2).
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Endometrium
The endometrial response to estrogen administration was restricted to an increase in the endothelial NOS isoform. Expression of type I NOS was not significantly increased by either 5 or 8 days of estrogen replacement. Although a trend toward an increase with both replacement regimes was observed for type I NOS mRNA levels (Fig. 3, left panel), there was no noticeable change in protein mass (Fig. 3, right panel). In the case of type III NOS expression, a significant increase (4-fold, p < 0.05) in mRNA was observed after the 5-day administration (Fig. 3 left panel, Tukey's test). The concomitant increase in type III NOS protein mass was only 1.5-fold (Fig. 3, right panel). The longer estradiol replacement regime was associated with an increased variability in the endometrial response. The magnitude of the response in type III NOS mRNA and protein mass was smaller and showed greater variability in the 8-day treatment group compared to the 5-day group and was not statistically different from control (Tukey's test, Fig. 3).
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Caruncular Endometrium
This uterine compartment exhibited the highest levels of type I NOS mRNA and protein mass of all three uterine compartments. As shown in Figure 4, high levels of mRNA were present in caruncles of ovariectomized control animals. Notwithstanding the high baseline levels, both estrogen replacement regimes significantly increased type I steady-state mRNA levels (p < 0.05, Tukey's test). Type I NOS protein mass was also increased in the 5-day and 8-day estrogen replacement groups (p < 0.05 Tukey's test; Fig. 4, right panel). However, the magnitude of the increase in protein mass was considerably higher than the change in steady-state mRNA levels: to 1.3- and 1.5-fold for the change in mRNA vs. 4.9- and 5.6-fold for the change in protein after 5 or 8 days of estrogen replacement, respectively. Despite the absence of significant changes in type III NOS mRNA expression with either estrogen replacement, type III NOS protein mass was significantly increased in the 5-day estrogen replacement group (p < 0.05, Tukey's test). Similar to the endometrium response, the effect of estrogen on type III NOS protein mass was no longer present in the 8-day estrogen administration group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Estrogen has clear beneficial effects on the cardiovascular system; however, controversy still exists regarding the relative role of NOS in these effects. Notwithstanding several studies reporting a stimulatory effect of estrogen on type III NOS expression both in vivo and in vitro [18–25], the central issue in the controversy is the absence of a canonical estrogen response element (ERE) in type III NOS. The type III NOS gene contains several repeats of the 5' half of the ERE palindrome, and an ERE-like sequence. These sequences, however, do not bind to activated estrogen receptors [26]. Estrogen increases the expression and/or activity of type III NOS in vascular endothelium by genomic and nongenomic mechanisms [22, 27]. In vitro, estrogen has a biphasic effect on type III NOS expression. Initially, estrogen increases type III NOS activity in a receptor-mediated manner that does not require synthesis of mRNA or new protein [28]. In fetal sheep pulmonary artery endothelial cells, this effect has a latency of 5 min [22, 28], and a similar effect is present in female human umbilical vein endothelial cells (HUVEC) [23]. In both cell types, this acute effect is followed by an increase in type III NOS mRNA at 48 h. Most likely the mechanism for the estrogen-induced increase in type III NOS mRNA is not mediated by a direct interaction between the estrogen receptor-ERE complex and regulatory sites of the NOS gene, but indirectly through interaction of the NOS gene with a transcription factor increased by estrogen. Increased binding activity or concentration of a transcription factor, i.e., AP-1, Sp-1, NF-1, or NF-
B, has been suggested as one such mechanism on the basis of compelling evidence for the involvement of Sp-1 in the in vitro response to estrogen in a human endothelial cell line [26]. In contrast, no information exists on the mechanisms by which estrogen stimulates the expression of type I NOS in central nervous system tissue and skeletal muscle [16, 17]. The gene for type I NOS also lacks the classical ERE, and by analogy to the case of type III NOS, the effect of estrogen on type I NOS expression probably does not involve binding of the activated estrogen receptor to regulatory sequences in the type I NOS gene. Theoretically, the measured increase in NOS mRNA and protein could have been determined by cell proliferation as opposed to an increase in gene expression. Although one of the effects of estrogen on the sheep uterus is cell proliferation, the increase in DNA content is not proportional to the increase in organ size, indicating that the predominant effect is cell hypertrophy [29]. Recently, the same authors have shown that in response to estrogen, there is a marked proliferation in endometrial vascular tissue [30]. Therefore, we have interpreted our data on type I NOS to most likely represent an increase in gene expression. The changes in type III NOS expression, however, could be in part a reflection of endothelial cell proliferation.
In the uterus, the reported effects of estrogen on NOS activity and/or expression vary with the species and the particular isoform studied. In rats and rabbits, chronic estrogen replacement decreases NOS activity in the uterine horns [12, 13]. In contrast, estrogen administration to mice and sheep increases NOS [9, 14]. Our data show that the effects of estrogen on NOS expression within the uterus also depend to a great extent on the type of tissue being stimulated. We clearly show a differential effect of estrogen on type I NOS and type III NOS expression in the different compartments of the nonpregnant sheep uterus. Estrogen increases type I NOS mRNA and protein mass in myometrium and caruncles but not in glandular endometrium. The amount of type I NOS mass present in myometrium and endometrium of control sheep was found to be very similar (Fig. 1). However, type I NOS expression in endometrium was unaffected by either the 5-day or the 8-day estrogen treatment. Interestingly, the caruncle (nonglandular endometrium) exhibited a pronounced response to estrogen, and the magnitude of the change in NOS protein was slightly larger than the myometrial response. The effect of estrogen on type III NOS expression within the uterus was also shown to be dependent on the tissue type. A coordinated increase in type III NOS mRNA and protein was evident after 5 days of estrogen replacement only in endometrium, and this effect was lost after 8 days of estrogen. The decrease in the type III NOS expression response after prolonged estrogen administration correlates very well with the in vivo effects of estrogen on uterine blood flow. In nonpregnant sheep, the increase in uterine blood flow in response to continuously administered estrogen peaks at 3 days and within 5 days is back to baseline, whereas the effect of estrogen on cardiac output was maintained for at least 2 wk [31]. Magness et al. [32] have recently reported that the magnitude of the blood flow increase is approximately the same in the three uterine compartments after acute estrogen administration, but not during chronic estrogen administration. During prolonged estrogen administration, myometrial blood flow increases at the expense of caruncular flow [32], providing an excellent functional correlate to our findings that show an increase in type III NOS expression in myometrium after 8 days of estrogen replacement. The absence of desensitization to the effect of estrogen in the response of type I NOS expression in myometrium and caruncular endometrium suggests that the mediator of the estrogenic effect is not the same that regulates type III NOS expression and uterine blood flow. The AP-1 transcription factor is a potential candidate for the regulation of type I NOS in myometrium. This factor binds as a heterodimer of c-fos/c-jun. Estrogen increases c-jun in myometrium [33] and decreases c-jun expression [33–35] in endometrium.
The functional relevance of the changes in uterine type I NOS is not very clear. Myometrial type I NOS has a diffuse distribution in the myometrial smooth muscle, and it is found as a soluble cytoplasmatic enzyme and also as a membrane-bound enzyme. Administration of the NOS inhibitor L-NAME to nonpregnant sheep does not increase uterine contractility, despite a marked increase in arterial blood pressure; thus the role for NOS as a modulator of uterine contractility is unlikely [36]. The physiological role for type I NOS in caruncular endometrium is even less clear. Although type I NOS in myometrium and caruncles could participate in the regulation of uterine blood flow on the basis of the temporal profile of the expression pattern in response to estrogen administration, this possibility seems unlikely because type I NOS expression is high at a time when uterine blood flow is preferentially shunted to the myometrium [32]. Recently, evidence has accumulated indicating that NOS can affect glucose metabolism in skeletal muscle. The effect of NO can occur independently of insulin and also as a modulator of the effects of insulin [37, 38]. NO regulates glucose metabolism by increasing glucose transport, glucose utilization, and oxidative metabolism [38–40]. Estrogen has profound effects on uterine glucose metabolism, also in a uterine compartment-specific manner [41, 42]. Thus, it is possible that some of the effects of estrogen are mediated by an increase in NO production by type I NOS. Glucose utilization by uterine strips obtained from estrogen-treated ovariectomized rats increases when the strips are exposed to the NO donor sodium nitroprusside, and it is decreased by high concentrations of NOS inhibitors [43].
In summary, our data show that in the uterus of the nonpregnant ovariectomized sheep, the expression of the neuronal and endothelial NOS isoforms increase in a tissue compartment-specific manner in response to estrogen replacement. The presence of type I NOS in caruncles, a noncontractile tissue, suggests that the physiological role for NO in the uterus is not only that of a smooth muscle relaxant. The role of NO as a regulator of myometrial glucose utilization requires further investigation.
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FOOTNOTES |
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2 Correspondence: Jorge P. Figueroa, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. FAX: 336 716 6937; figueroa{at}wfubmc.edu
Accepted: December 15, 1998.
Received: August 21, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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