Differential expression of uterine NO in pregnant and nonpregnant rats with intrauterine bacterial infection

Previous studies have demonstrated that nitric oxide (NO) is involved in the uterine host defense against bacterial infection. In nonpregnant rats, NO production in the uterus was shown to be lower, and inducible NO synthase (NOS) expression was undetectable. However, studies in pregnant rats show abundant expression of inducible NOS with significant elevation in NO production in the uterus. We have recently reported that intrauterine Escherichia coli infection caused a localized increase in uterine NO production and inducible NOS expression in the nonpregnant rat. In our present study, we examined whether the uterine NO production, NOS expression, and uterine tumor necrosis factor-alpha protein are increased in pregnant rats with intrauterine pathogenic Escherichia coli infection. Unlike the nonpregnant state, the NO production in the infected uterine horn of pregnant rats was not significantly elevated after bacterial inoculation compared with the contralateral uterine horn. The expression of uterine NOS (types II and III) also did not show significant upregulation in the infected horn. This is in contrast to that in nonpregnant animals, in which type II NOS was induced in the uterus on infection. Moreover, intrauterine infection induced an elevated expression of tumor necrosis factor-alpha protein in the infected horn both of nonpregnant and of pregnant rats. These data suggest that the sequential stimulation of NOS expression, especially the inducible isoform, and generation of uterine NO are lacking during pregnancy despite an elevated tumor necrosis factor-alpha after infection. In summary, NO synthesis response may be maximal at pregnancy, and infection may not further induce the NO system. Present studies, together with our previous report that intrauterine infection-induced lethality in pregnancy rats was amplified with the inhibition of NO, suggest that pregnancy is a state predisposed for increased complications associated with intrauterine infection and that the constitutively elevated uterine NO during pregnancy may help contain or even reduce the risk of infection-related complications.     

Impact of the estrus cycle and reduction in estrogen levels with aromatase inhibition, on renal function and nitric oxide activity in female rats

Estradiol increases mRNA and/or protein expression of the nitric oxide synthase (NOS) isoforms in a variety of tissues including kidney. In this study we determined the relationship between cyclical variations in estradiol levels and renal function and total NO production in the virgin female rat. In addition, we used an aromatase inhibitor (Anastrozole), to inhibit synthesis of estradiol from testosterone. Estradiol levels were higher in proestrus vs. diestrus, and were markedly suppressed by 7 days treatment with aromatase inhibitor. There was no difference in total NO production (from urinary and plasma nitrate + nitrite = NO_X) between proestrus and diestrus but aromatase inhibition resulted in increases in total NO production. The renal cortical NOS activity and protein abundance also increased in aromatase-inhibited female rats. There were no differences in blood pressure (BP) in any group but the renal vascular resistance (RVR) was low in proestrus, increased in diestrus and did not change further after aromatase inhibition. In summary, the cyclical changes in renal function correlate with estradiol but not NO levels. Pharmacologic castration with aromatase inhibition leads to a marked increase in total and renal NOS. This contrasts to earlier work where surgical castration causes decreased NOS.     

Regulation of thioredoxin mRNA in the rat uterus by gonadal steroids

Estradiol has been shown to increase the level of thioredoxin mRNA in the uterus of the ovariectomized (ovx) rat. In this study the influence of progesterone, androgens, the anti-estrogen ICI 182780 and the anti-androgen Flutamid on thioredoxin expression, has been studied in the rat uterus. Thioredoxin mRNA concentrations were determined by solution hybridization. Ovx rats treated with progesterone alone showed no effect on thioredoxin expression. Combined treatment of ICI 182780 and estradiol attenuated the estradiol-induced increase in thioredoxin mRNA. When ovx rats were treated with a testosterone depot, the amount of thioredoxin mRNA was increased five-fold after 48 h and remained at that level during the rest of the 168 h monitored. A similar increase in thioredoxin mRNA could be seen after 5-dihydrotestosterone treatment, indicating a true androgenic effect. In addition, the anti-androgen Flutamid attenuated the thioredoxin mRNA increase seen after 5-dihydrotestosterone treatment alone.

It is concluded that thioredoxin mRNA is regulated by growth promoting gonadal steroids in the rat uterus. The attenuation of the estrogen and androgen-induced increases of the thioredoxin mRNA with ICI 182780 and Flutamid, indicate that the effect is mediated via the estrogen receptor and androgen receptor respectively. None of these hormones affected the hepatic thioredoxin mRNA level in the same animals.     

Quantitative nitric oxide production by rat, bovine and porcine macrophages

The aim of this work was to compare in vitro nitric oxide (NO) production by rat, bovine and porcine macrophages. NO production was induced by lipopolysaccharide (LPS) or by phorbol 12-myristate 13-acetate (PMA) with ionomycin or recombinant interferon gamma (rIFN-γ) and was assessed by Griess reaction. NO synthase type II (NOS II) expression was quantified by immunocytochemistry, Western blot and real-time polymerase chain reaction (RT-PCR). There were differences in NO production by pulmonary alveolar macrophages (PAM) in all species tested. The largest amounts of NO were produced by rat PAM. Less NO was produced by bovine PAM. Moreover, PAM in rats and cows differed in their abilities to respond to various stimulators. Neither porcine PAM nor Kupffer cells produced NO. Stimulation of porcine PAM with alternative concentrations of LPS did not lead to inducing NO production. Stimulation of porcine PAM with rIFN-γ together with LPS led to a significant increase in the expression of NOS II mRNA, albeit without detectable NO production or NOS II expression on the protein level.     

Estrogen regulation of c-fos messenger ribonucleic acid

Acute administration of 17 beta-estradiol to immature female rats elicits a rapid and striking increase in the size of the uterus. This increase in size to caused by both hypertrophy and hyperplasia in the epithelial, stromal, and myometrial cells in the uterus. Previous studies have shown that induction of mRNA for the epidermal growth factor receptor, the cellular homolog of the erb-B oncogene, occurs early during estrogen-stimulated uterine growth. We report here that estradiol causes a very rapid induction of the mRNA for the cellular oncogene c-fos in immature rat uterus. Steady state levels of c-fos mRNA reach a maximum 3 h after 17 beta-estradiol administration and slowly return to low basal levels in 15 h. Dexamethasone, progesterone, and 5 alpha-dihydrotestosterone had no effect on uterine c-fos mRNA expression. The induction of c-fos mRNA by estrogen was unaffected by the protein synthesis inhibitor puromycin but completely abolished by the RNA synthesis inhibitor actinomycin D.     

Vasorelaxant action of 17 -estradiol in rat uterine arteries: role of nitric oxide synthases and estrogen receptors

The uterine vasculature plays an important role during pregnancy by providing adequate perfusion of the maternal-fetal interface. To this end, substantial remodeling of the uterine vasculature occurs with consequent changes in responsiveness to contractile agents. The purpose of our study was to characterize the vasorelaxant effects of estrogens on vascular smooth muscles of the rat uterine artery during pregnancy and to evaluate the involvement of estrogen receptors (ESR) and nitric oxide synthases (NOS). To do so, we measured NOS expression in the whole uterine and mesenteric circulatory bed by Western blotting. Vasorelaxant effects of 17beta-estradiol (17beta-E(2)) were assessed on endothelium-denuded uterine arteries with wire myographs in the absence and presence of pharmacological modulators [nitro-L-arginine methyl ester (L-NAME), ICI-182780, tamoxifen]. All experiments were performed on arteries from nonpregnant (NP) and late pregnant (P) rats. In the uterine vasculature of the latter group, NOS3 (endothelial NOS) expression was increased, while NOS1 (neuronal NOS) was reduced compared with NP rats. Expression of the NOS2 (inducible NOS) isoform was undetectable in the two groups. Both 17beta-E(2) and 17alpha-E(2) induced uterine artery relaxation, but the latter evoked lower responses. Endothelium-denuded arteries from NP rats showed larger relaxation with 17beta-E(2) than P rats. This larger relaxation disappeared in the presence of L-NAME. The ESR antagonist ICI-182780 did not affect acute relaxation with 17beta-E(2) and 17alpha-E(2). Moreover, membrane-nonpermeant 17beta-E(2):BSA (estradiol conjugated to bovine serum albumin) did not induce any vasorelaxation. Our results indicate that estrogens exert direct acute vasorelaxant effects in smooth muscles of the rat uterine artery that are mediated by mechanisms independent of ESR activation, but with some stereospecificity. Part of this effect, in NP rats only, is due to nitric oxide produced from muscle NOS1.     

Alterations in gene expressions encoding preproET-1 and NOS in pulmonary tissue in endotoxemic rats

Septic shock is characterized by hypotension and a hyporeactive response to vasopressor agents. The pathogenesis is due to vascular leaks and an increased synthesis of cytokines and nitric oxide (NO). The present study examined the time-dependent alterations of endothelin-1 (ET-1) and the expression of NO synthase (NOS) in lung tissue in a septic rat model. Normal Sprague-Dawley (SD) rats aged 10 weeks received 15 mg/kg lipopolysaccharide (LPS) and then were sacrificed at different time points (1, 3, 6, and 10 hrs). Rats that did not receive LPS were considered to be controls. Both systolic and diastolic pressure decreased in SD rats after LPS administration. Time-dependent onset of features of acute lung injury, such as the infiltration of inflammatory cells and thickening of alveolar septa, were seen in rats that received LPS. A 2.8-fold increase in the expression of preproET-1 level was observed in lung tissue 6 hrs after LPS administration. The expression of endothelial NOS (eNOS) was also altered in lung tissue in a time-dependent fashion. After the administration of LPS, there was a 16-fold increase in the expression of eNOS mRNA. The peak expression of inducible NOS (iNOS) in lung tissue specimens obtained from rats that received LPS was 45-fold higher than that in control rats. ET-1 is a potent vasoconstrictor and thereby may play an important role in the pathogenesis of acute lung injury in a septic rat model. The increased expression of NOS may result in excess NO production and may also play a role in the pulmonary complications of endotoxemia.     

尾加压素对新生大鼠心肌细胞一氧化氮合成的影响

应用半定量逆转录－多聚酶链反应法,观察尾加压素（urotensin Ⅱ,UⅡ）对培养的新生SD大鼠心肌细胞内皮型一氧化氮合酶（endothelial nitric oxide synthase,eNOS)mRNA表达的影响,并测定UⅡ对心肌细胞内一氧化氮合酶（nitric oxide synthase,NOS)活性和一氧化氮（nitric oxide,NO)释放的影响。结果显示：UⅡ抑制培养的新生大鼠心肌细胞eNOS mRNA表达、抑制NOS的活性及NO释放;0.1μmol/L浓度的UⅡ呈时间依赖性抑制心肌细胞NOS的活性及NO生成。上述实验结果提示UⅡ的心血管作用可能与NO合成系统有关。     

Role of the nongravid part of the uterus in the luteolytic effects of estrogen in pregnant rats

Effects of a low dose of estradiol on the luteal function were studied in intact pregnant rats. The pregnant rats received daily sc injections of 0.1 microgram estradiol or sesame oil from day 7 to 14 of pregnancy (day 1 = day of insemination). Serum progesterone levels on day 15 were significantly lower in the estrogen-treated group than in the oil-treated group. In order to study how estrogen induced luteolysis, the pregnant rats received each of the following treatments on day 7 of pregnancy: (1) The uterus except that containing a single conceptus was removed by hysterectomy (hysterectomy group); (2) All but a single conceptus were removed by aspiration, so that rats carried only a single conceptus with the whole part of the nongravid uterus (aspiration group). Each group of rats received also sc injections of 0.1 microgram estradiol or sesame oil from day 7 to 14 of pregnancy. Estradiol treatment caused a significant decline in serum progesterone levels in the aspiration group on day 15, but this was not the case in the hysterectomy group. There was no significant difference in serum LH levels among any of the groups on day 15 of pregnancy. These results indicated that estradiol induced luteolysis in the intact pregnant rats, which would, at least in part, be mediated through the uterus.     

氧化修饰低密度脂蛋白对巨噬细胞一氧化氮合酶基因表达的影响

氧化修饰LDL(OX-LDL)可抑制脂多糖(LPS)诱导的巨噬细胞NO释放, 而正常(N-LDL)和乙酰化LDL(AC-LDL)则没有抑制作用.OX-LDL对NO释放的抑制作用随LDL修饰程度的升高而增强,且具有浓度和时间效应.狭缝杂交结果显示OX-LDL处理可使LPS诱导的巨噬细胞NOS mRNA含量下降,提示OX-LDL对NO释放的抑制作用可能发生在转录水平.     

金雀异黄酮通过减少卵巢切除大鼠心肌小凹蛋白-1的表达而增加一氧化氮的生成

观察金雀异黄酮(genistein)替代治疗对卵巢切除大鼠心肌中一氧化氮(nitric oxide,NO)和内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)的影响.成年雌性Sprague-Dawley大鼠经双侧卵巢切除术,假手术组作为对照,术后三周将行卵巢切除术的大鼠随机分为低剂量genistein(0.5 mg/kg·d1)、高剂量genistein(5.0 mg/kg·d-1)、17-β雌二醇(0.1 mg/kg·d-1)和模型组(100μl/d芝麻油),各组均皮下注射给药并给予不含大豆的饲料喂养6周,测定大鼠尾动脉血压、心率,麻醉后放血处死大鼠称量子宫重量;放免法检测血浆中总雌二醇,亚硝酸还原酶法检测心肌匀浆中NO,Western blot检测心肌中eNOS的表达以及eNOS的调节蛋白小凹蛋白-1(caveolin-1)和钙调素(calmodulin)的表达情况.结果显示各组间大鼠血压无显著性差异,同17-β雌二醇一样,genistein能呈剂量依赖性地增加心肌组织中eNOS表达量和NO生成,同时genistein能明显降低内源性eNOS活性抑制物caveolin-1的表达,而不影响eNOS活性正性调节蛋白钙调素的表达.与溶媒对照组比较,0.5 mg/kg·d-1的genistein不增加子宫重量,5.0 mg/kg·d-1的genistein增加子宫重量3倍,但较17-β雌二醇(增加6倍)的作用小(P＜0.01).上述结果提示,植物雌激素genistein剂量依赖性地上调心肌组织eNOS的活性并增加NO的生成,减少抑制eNOS活性的小凹蛋白-1表达.     

Effect of estradiol on estrogen receptor expression in rat uterine cell types

In rodent uterus, both up- and down-regulation of estrogen receptor alpha (ERalpha) messenger ribonucleic acid (mRNA) and protein levels by estradiol has been demonstrated; however, it is not known which of the uterine compartments (endometrial epithelium, stroma, myometrium) respond to estradiol with autoregulation of ERalpha. The purpose of the present study was to investigate and compare the kinetics and cell type-specific effects of estradiol on uterine ERalpha expression in immature and adult rats. Ovariectomized female rats were injected s.c. with sesame oil or estradiol-17beta. Uteri were collected and analyzed for changes in ERalpha mRNA using RNase protection assays (RPA) and in situ hybridization using radiolabeled probes specific for ERalpha. Immunohistochemical analysis was performed with a polyclonal antibody specific to ERalpha. Expression of ERalpha in the uterine epithelial cells decreased at 3 and 6 h after estradiol administration to immature and adult rats, respectively. At 24 h, ERalpha mRNA levels in the immature and mature rat uterus were higher than pretreatment levels but returned to baseline by 72 h. Pretreatment with cycloheximide did not block the 3-h repressive effect of estradiol, suggesting that the estradiol-induced decrease in ERalpha mRNA occurs independent of new protein synthesis. A decrease in ERalpha mRNA and protein was also observed in uterine epithelia at 3 and 6 h after an estradiol injection to immature and adult rats, and intensity of both the in situ hybridization signal and the immunostaining in the epithelium increased at 24 and 72 h. However, the periluminal stromal cells in the adult uterus and the majority of stromal cells of the immature uterus appeared to have increased ERalpha expression. The results indicate that down-regulation of ERalpha in the epithelia and up-regulation of stromal ERalpha play a role in early events associated with estradiol-induced cell proliferation of the uterine epithelia.     

Regulation of insulin-like growth factor-I and thioredoxin expression by estradiol in the reproductive tract of the prepubertal female lamb

Estradiol (E(2)) has been shown to be an important uterine growth promoting molecule in the ovariectomized (ovx) rat, which increases the mRNA levels of insulin-like growth factor-I (IGF-I) and the redox enzyme thioredoxin. The aim of this study was to explore the role of E(2) in the regulation of IGF-I and thioredoxin in the reproductive tract of the prepubertal female lamb. Twenty 3-month-old lambs were treated with i.m. injections of E(2) at 24 h intervals. The animals were sacrificed 12 or 24 h after the last injection, and 72 h was the longest treatment period. The mRNA levels of thioredoxin and IGF-I were determined by a solution hybridization technique. There was a 5-fold increase in the cervical IGF-I mRNA level 12 h after the first E(2) injection. The uterine IGF-I mRNA level was doubled after 12 h and this increase was maintained during the rest of the experimental period. The IGF-I mRNA level in the oviducts was more than doubled 12 and 24 h after the E(2) injection, then the level decreased towards the initial level. The thioredoxin mRNA level in the cervix was increased 4-fold after 24 h, whereas no significant effect was seen in the uterus. The thioredoxin mRNA level in the oviduct was more than doubled 12 and 24 h after the first E(2) injection. Thus, estradiol regulates the expression of IGF-I and thioredoxin in the reproductive tract of prepubertal lambs.     

Nonpregnant sheep uterine type I and type III nitric oxide synthase expression is differentially regulated by estrogen

The aim of this study was to characterize the effect of estrogen on the expression of neuronal and endothelial isoforms of nitric oxide (NO) synthase (NOS) in myometrium, endometrium, and caruncle (nonglandular endometrium) in nonpregnant sheep. Twenty sheep were castrated during synchronized estrus (Days 14-16) and 4 days after surgery treated i.v. through the jugular with 100 microg/day of estradiol-17beta for 5 (n = 6) or 8 (n = 6) days or with vehicle (n = 8). Nitric oxide synthase mRNA was measured by ribonuclease protection assay, and NOS protein mass was measured by Western immunoblotting. Data were analyzed by ANOVA and Tukey's test. The three distinct uterine compartments studied contained the mRNA and protein for the neuronal (type I NOS) and the endothelial (type III NOS) isoforms of NOS. However, no inducible NOS was detected. Estrogen exhibited a differential effect on NOS expression in a tissue compartment- and NOS isoform-specific manner. In myometrium and caruncles, but not in endometrium, type I NOS mRNA and protein mass increased significantly (p < 0.05) after 5 or 8 days of estrogen. In contrast, type III NOS increased significantly in myometrium only after 8 days, whereas in endometrium and caruncles the increase was significant in the 5-day treatment group (p < 0.05). We conclude that the expression of type I NOS and type III NOS in the uterus are differentially regulated by estrogen. This differential regulation suggests that the NO produced within the uterus serves more than one physiological role. In myometrium it may be a uterorelaxant and regulate glucose utilization, and in endometrium and myometrium it may regulate blood flow.     

The role of nitric oxide in the effects of ovarian steroids on spontaneous myometrial contractility in rats

Forty ovariectomized rats were apportioned into one control and three experimental groups (n=10 each) to evaluate the role of nitric oxide in the effects of ovarian steroids on spontaneous myometrial contractility in rats. The control group (group Ov) received sesame oil once daily for 10 days, whereas rats in the experimental groups were treated with progesterone (2 mg/(rat day); group P), 17beta-estradiol (10 microg/(rat day); group E2), or progesterone and 17beta-estradiol together (group E2+P). The functionality of the arginine-nitric oxide synthase (NOS)-nitric oxide (NO) pathway in the uterine horns of sacrificed rats was evaluated in an isolated organ bath. L-Arginine, sodium nitroprusside (SNP) and 8-Br-cGMP decreased uterine contractile tension induced by electric field stimulation (EFS) in the Ov, P, and E2+P groups, but not in the E2 group. In addition, L-arginine was ineffective when applied together with a NOS inhibitor, L-nitro-N-arginine (L-NNA). The percentage of contractile inhibition was higher in the Ov and P groups compared to the E2+P group. Immunohistochemical evaluation revealed that expression of neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) in smooth muscles and nerve cells did not differ among the groups. Expression of nNOS and eNOS was strongly evident in the E2 and E2+P groups at both surface and glandular epithelium of the endometrium. iNOS expression was increased in surface epithelium of the E2 and E2+P groups. However, iNOS expression was only increased in glandular epithelial cells of the E2+P group. In conclusion, the L-arginine-NOS-NO pathway inhibits myometrial contractions via cGMP-dependent and -independent mechanisms, and while progesterone maintains the nitric oxide effects, estrogen prevents them. These results suggest that NOS does not mediate the effects of estrogen.     

Effect of estradiol on the amino acid-accepting activity of uterine tRNAs and their participation in protein synthesis

Estradiol (E2) induces a complementary increase in both the amount of mRNA and the rate of translation of the mRNA in the uterus of ovariectomized mature rats. The mechanism of the translational effect was evaluated by measuring the functional capacity of uterine tRNA isolated from control, E2 (1 h)- and E2 (14 h)-treated ovariectomized rats to support amino acid acceptor activity and uterine protein synthesis. The specific amino acid acceptor activity (SAA) of deacylated tRNA for 18 individual amino acids was determined using a tRNA-dependent rat liver tRNA synthetase preparation. The SAA was the same for all amino acids for uterine tRNA from control and E2 (1 h)-treated rats but was increased for uterine tRNA from E2 (14 h)-treated rats to levels that were 1.4-4.3 times the SAA of uterine tRNA from control rats. When uterine tRNA from control and E2 (14 h)-treated rats was incubated with purified tRNA nucleotidyltransferase, the SAA for all amino acids was increased an average of 1.6-fold for control tRNA and 0.3-fold for tRNA from E2 (14 h)-treated rats. The ability of uterine tRNA to support maximal rates of protein synthesis in tRNA-dependent uterine ribosome protein synthesis assay was increased by either in vivo treatment of the rats with estradiol or by in vitro repair of the 3'-CCA terminus of this tRNA by nucleotidyltransferase. These observations suggest that E2 may increase the rate of mRNA translation in the uterus, in part, by increasing the proportion of certain tRNAs with intact and functional 3'-CCA acceptor termini.