Different role of endothelium/nitric oxide in 17beta-estradiol- and progesterone-induced relaxation in rat arteries

The present study was aimed to examine the different role of endothelium/nitric oxide in relaxation induced by two female sex hormones, 17beta-estradiol and progesterone in rat isolated aortas and mesenteric arteries. The isometric force of each ring was measured with Grass force-displacement transducers in the organ bathes. 17beta-Estradiol induced both endothelium-dependent and -independent relaxation in the rat aortas but only the endothelium-independent relaxation in the rat mesenteric arteries. In contrast. progesterone induced both endothelium-dependent and -independent relaxation in the rat mesenteric arteries but only endothelium-independent relaxation in rat aortas. N(G)-Nitro-L-arginine methyl ester and methylene blue attenuated the relaxant response to 17beta-estradiol in the aortic rings or to progesterone in the mesenteric arteries. Pretreatment with L-arginine antagonized the effect of N(G)-nitro-L-arginine methyl ester on sex hormone-induced relaxation. The endothelium contribution to relaxation seems to only relate to lower concentrations of 17beta-estradiol and progesterone. In summary, the present results clearly demonstrate a different role of the functional endothelium in the relaxant response to 17beta-estradiol or progesterone in the conduit vessel (aorta) and the resistance vessels (mesenteric artery). Nitric oxide contributes largely to the endothelium-dependent relaxation induced by 17beta-estradiol in the isolated aortas or by progesterone in the mesenteric arteries.     

17β-雌二醇诱导血管内皮细胞一氧化氮释放及其与细胞内钙的关系

利用小牛胸主动脉内皮细胞（ＢＡＥＣｓ）作为模型,观察１７β－雌二醇（Ｅ２）ＢＡＥＣｓ一氧化氮（ＮＯ）释放、一氧化氮合酶（ｅＮＯＳ）ｍＲＮＡ表达和细胞内钙（〔Ｃａ＾２＋〕ｉ）的影响,以及雌激素受体（ＥＲ）拮抗剂ｔａｍｏｘｉｆｅｎ和ＮＯＳ抑制剂（Ｌ－ＮＡＭＥ）的作用。结果显示,Ｅ２（１０＾－１２￣１０＾－８ｍｏｌ／Ｌ）呈尝试依赖性促进ＢＡＥＣｓ中ＮＯ的释放,以１０＾－８ｍｏｌ／Ｌ浓度Ｅ２处理ＢＡＥＣｓ     

Multimodality imaging of nitric oxide and nitric oxide synthases

Nitric oxide (NO) and NO synthases (NOSs) are crucial factors in many pathophysiological processes such as inflammation, vascular/neurological function, and many types of cancer. Noninvasive imaging of NO or NOS can provide new insights in understanding these diseases and facilitate the development of novel therapeutic strategies. In this review, we will summarize the current state-of-the-art multimodality imaging in detecting NO and NOSs, including optical (fluorescence, chemiluminescence, and bioluminescence), electron paramagnetic resonance (EPR), magnetic resonance (MR), and positron emission tomography (PET). With continued effort over the last several years, these noninvasive imaging techniques can now reveal the biodistribution of NO or NOS in living subjects with high fidelity which will greatly facilitate scientists/clinicians in the development of new drugs and/or patient management. Lastly, we will also discuss future directions/applications of NO/NOS imaging. Successful development of novel NO/NOS imaging agents with optimal in vivo stability and desirable pharmacokinetics for clinical translation will enable the maximum benefit in patient management.     

Nongenomic action of progesterone in rat aorta: role of nitric oxide and prostaglandins

The mechanism of action of progesterone (Pg) on rat vascular tissue was investigated. We obtained evidence that 10-nM Pg inhibited platelet aggregation at 1-5 min. Previously, we reported that nitric oxide (NO) mediated this antiaggregatory effect. Rat aortic strips (RAS) NO synthase (NOS) activity in response to "in vitro" treatment with other sex steroids hormones was measured. The stimulatory action of Pg on NO production was specific for ovarian hormones and depends on sex. The effect was nongenomic since cycloheximide did not suppress the increment in NO induced by Pg. Finally, we demonstrated that Pg (5 min) increased prostacyclin release (42-182% above control) in a dose-dependent manner (1-100 nM). Indeed, indomethacin (10 microM) completely suppressed the increment in citrulline levels induced by the hormone. These results suggest that Pg exerts a direct nongenomic action on rat aortic metabolism, which involves NOS and cyclooxygenase (COX) activation and a cross-talk between NO- and prostacyclin (PGI(2))-dependent pathways.     

Autonomic regulation of pacemaker activity: role of heart nitric oxide synthases

In autonomic-blocked rats treated with NG-nitro-L-arginine methyl ester (L-NAME, 7.5 mg/kg), heart rate increased 18% and mean arterial pressure increased 48%. Thyroidectomy, along with autonomic blockade, hampered the chronotropic response but did not modify the effect on blood pressure. After 150 min of autonomic blockade, the experimental end point, total nitric oxide (NO) production by heart NO synthases (NOS) decreased 61%: from 54 to 21 nmol NO.min-1.g heart-1. Mitochondrial NOS (mtNOS) and sarcoplasmic reticulum endothelial NOS activities decreased 74% and 52%, respectively. Mitochondria isolated from whole heart showed a well-coupled oxidative phosphorylation with high respiratory control and ADP-to-O ratios, decreased mtNOS activity (55-60%), and decreased mtNOS protein expression (70%). Immunohistochemistry with anti-inducible NOS antibody linked to gold particles localized mtNOS at the inner mitochondrial membranes. Histochemical right atrial NOS (NADPH-diaphorase) decreased 55% after heart denervation. The effects of autonomic denervation on the NO system were partially prevented by thyroidectomy performed simultaneously with autonomic blockade. Western blot analysis indicated a very rapid mtNOS protein turnover (half time=120 min) with a process of protein expression that was upregulated by thyroidectomy and a degradation process that was downregulated by the autonomic nervous system. The observations suggest that NO-mediated pathways contribute to pacemaker heart activity, likely through the NO steady-state levels in the right atrium and the whole heart.     

The role of nitric oxide in the metabolism of labeled glucose in isolated rat uterus. Influence of 17β-estradiol

The influence of nitric oxide (NO) on the production of ¹⁴CO₂ from labeled glucose in uteri isolated from ovariectomized-estrogenized rats was studied. Nitroprusside, an NO donor (NP), 200 μM increased the formation of labeled CO₂ from [U-¹⁴C]glucose. This effect was blunted by hemoglobin (Hb) 20 μg/mL, an NO scavenger. The addition of N-monomethyl arginine (NMMA), an inhibitor of NO synthase decreased the stimulatory action of NP at 400 mM. Incubation of uterine strips in the presence of NP plus acetylsalicylic acid (ASA) 10⁻⁴ M (a cyclooxygenase inhibitor), inhibited the stimulatory action of NP on glucose metabolism. PGE₂ (10⁻⁷ M) added to the incubation medium containing NP and ASA reversed the effect of the inhibitor. Neither NP nor Hb nor NMMA modified the ¹⁴CO₂ production from labeled glucose in uterine strips from ovariectomized rats. The addition of NP to the incubating medium increased PGE accumulation by uterine strips from rats treated with estradiol, but not in ovariectomized animals. These results suggest that NO exerts a positive influence on glucose metabolism and PGE synthesis in isolated rat uteri from estrogenized animals.     

The protein inhibitor of neuronal nitric oxide synthase (PIN): characterization of its action on pure nitric oxide synthases

Neuronal NO synthase (nNOS) was discovered recently to interact specifically with the protein PIN (protein inhibitor of nNOS) [Jaffrey, S.R. and Snyder, S.H. (1996) Science 274, 774–777]. We have studied the effects on pure NOS enzymes of the same GST-tagged PIN used in the original paper. Unexpectedly, all NOS isoenzymes were inhibited. The IC₅₀ for nNOS was 18±6 μM GST-PIN with 63 nM nNOS after 30 min at 37°C. Uncoupled NADPH oxidation was inhibited similarly, whereas cytochrome c reductase activity, the K_M for l-arginine, and dimerization were unaffected. We reconsider the physiological role of PIN in the light of these results.     

Pregnancy enhances G protein activation and nitric oxide release from uterine arteries

We hypothesized that pregnancy modulates receptor-mediated responses of the uterine artery (UA) by altering G protein activation or coupling. Relaxation and contraction to NaF (0.5-11.5 mM), acetylcholine (10(-9)-10(-5) M), and bradykinin (10(-12)-3 x 10(-5) M) were measured in isolated UA of pregnant and nonpregnant guinea pigs. Responses were measured in the presence and absence of either cholera toxin (2 microg/ml) or pertussis toxin (Galpha(s) and Galpha(i) inhibitors, respectively). NaF relaxation was endothelium dependent and nitro-L-arginine sensitive (a nitric oxide synthase inhibitor). Relaxation to NaF, acetylcholine, and bradykinin were potentiated by pregnancy. Cholera but not pertussis toxin increased relaxation to acetylcholine and bradykinin in UA from nonpregnant animals, had no effect in UA from pregnant animals, and abolished the pregnancy-induced differences in acetylcholine relaxation. Cholera toxin potentiated the bradykinin-induced contraction of UA of both pregnant and nonpregnant animals, whereas pertussis toxin inhibited contraction of UA from pregnant animals only. Therefore, pregnancy may enhance agonist-stimulated endothelium-dependent relaxation and bradykinin-induced contraction of UA by inhibiting GTPase activity or enhancing Galpha(s) but not Galpha(i) activation in pregnant animals. Thus the diverse effects of pregnancy on UA responsiveness may result from hormonal modulation of G proteins coupled to their specific receptors.     

Molecular evolution of nitric oxide synthases in metazoans

Nitric oxide synthases (NOS), the enzymes responsible for the NO synthesis, are present in all eukaryotes. Three isoforms (neuronal, inducible and endothelial), encoded by different loci, have been described in vertebrates, although the endothelial isoform seems to be restricted to tetrapods. In invertebrates, a variety of NOS isoforms have been variably annotated as "inducible" or "neuronal", while others lack precise annotation. We have performed an exhaustive collection of the available NOS amino-acid sequences in order to perform a phylogenetic analysis. We hypothesized that the NOS isoforms reported in vertebrates derive from 1) different invertebrate NOS, 2) a single invertebrate ancestral gene, through an event related to the double whole genomic duplication that occurred at the origin of vertebrates, and 3) the endothelial form of NOS appeared late in the evolution of vertebrates, after the split of tetrapods and fishes. Our molecular evolution analysis strongly supports the second scenario, the three vertebrate NOS isoforms derived from a single ancestral invertebrate gene. Thus, the diverse NOS isoforms in invertebrates can be explained by events of gene duplication, but their characterization as "inducible" or "neuronal" should only be justified by physiological features, since they are evolutionarily unrelated to the homonym isoforms of vertebrates.     

Gene and protein expressions of nitric oxide synthases in ischemia-reperfused peripheral nerve of the rat

Thisstudy examined mRNA and protein expressions of neuronal (nNOS),inducible (iNOS), and endothelial nitric oxide synthases (eNOS) inperipheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. Onesciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve werequantitatively measured by competitive PCR, and protein was determinedby Western blotting and immunohistochemical staining. The resultsshowed that, after ischemia (2 h), both nNOS and eNOS proteinexpressions decreased. After I/R (2 h of ischemia followed by3 h of reperfusion), expression of both nNOS and eNOS mRNA andprotein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal thedynamic expression of individual NOS isoforms during the course of I/Rinjury. An understanding of this modulation on a cellular and molecularlevel may lead to understanding the mechanisms of I/R injury and tomethods of ameliorating peripheral nerve injury.

     

Endothelial and inducible nitric oxide synthases in oocytes of cattle

Nitric oxide (NO) is a chemical messenger generated by the activity of the nitric oxide synthases (NOS). The NOS/NO system appears to be involved in oocyte maturation, but there are few studies on gene expression and protein activity in oocytes of cattle. The present study aimed to investigate gene expression and protein activity of NOS in immature and in vitro matured oocytes of cattle. The influence of pre-maturation culture with butyrolactone I in NOS gene expression was also assessed. The following experiments were performed: (1) detection of the endothelial (eNOS) and inducible (iNOS) isoforms in the ovary by immunohistochemistry; (2) detection of eNOS and iNOS in the oocytes before and after in vitro maturation (IVM) by immunofluorescence; (3) eNOS and iNOS mRNA and protein in immature and in vitro matured oocytes, with or without pre-maturation, by real time PCR and Western blotting, respectively; and (4) NOS activity in immature and in vitro matured oocytes by NADPH-diaphorase. eNOS and iNOS were detected in oocytes within all follicle categories (primary, secondary and tertiary), and other compartments of the ovary and in the cytoplasm of immature and in vitro matured oocytes. Amount of mRNA for both isoforms decreased after IVM, but was maintained after pre-maturation culture. The NOS protein was detected in immature (pre-mature or not) and was still detected in similar amount after pre-maturation and maturation for both isoforms. NOS activity was detected only in part of the immature oocytes. In conclusion, isoforms of NOS (eNOS and iNOS) are present in oocytes of cattle from early folliculogenesis up to maturation; in vitro maturation influences amount of mRNA and NOS activity.     

Analysis of rat uterine estrogen receptors by high-pressure liquid chromatography methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.