Expression of nitric oxide synthase protein and gene in the splanchnic organsof liver cirrhosis and portal hypertensive rats

AIM To investigate the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS)protein and eNOS mRNA gene in the splanchnic organs of liver cirrhosis and portal hypertensive rats.METHODS In control and CCl4-induced liver cirrhotic rats, the expression of eNOS and iNOS proteins wasdetected by immunohistochemical method, and eNOS mRNA was detected by in situ hybridization.RESULTS The expression of eNOS protein and eNOS mRNA increased in most organs of the cirrhotic rats,including bronchial and alveolar epithelial cells, renal tubular epithelial cells and mesenchyma, endothelialand adventitial cells of aorta and superior mesenteric artery, whereas no significant increase of iNOS proteinwas found. In the hepatic tissue, NOS protein and eNOS mRNA were present in mesenchymal cells and vesseladventitial cells, no difference was observed in the expression between control and cirrhotic rats.CONCLUSION The expression of NOS varied in region. In splanchnic organs and vasculars there was anincreased expression of eNOS which induced aplanchnic vasodilation and increased the inflow of portal vein,while in the liver tissue and blood vessel showed no increased expression, which may be associated withincreased intrahepatic vascular resistance.     

Hormonal and spatial regulation of nitric oxide synthases (NOS) (neuronal NOS, inducible NOS, and endothelial NOS) in the oviducts

Nitric oxide (NO) is a free radical produced by the action of NO synthases (NOS) and is known to be involved in the regulation of many reproductive events that occur in the oviducts. The oviducts are highly specialized organs that play crucial roles in reproduction by providing an optimal environment for the final maturation of gametes, fertilization, and early embryo development. In this study, we analyzed the expression, hormonal regulation, and cellular distribution of neuronal, inducible, and endothelial NOS in different bovine oviduct segments to better understand the roles played by these enzymes in oviductal functions in vivo. Quantitative RT-PCR analysis revealed that NOS isoforms are hormonally regulated and differentially expressed along the oviduct throughout the estrous cycle. All NOS were highly expressed around the time of estrus, and immunohistochemistry studies determined that neuronal NOS, inducible NOS (iNOS), and endothelial NOS are differentially distributed in cells along the oviduct. Interestingly, our results showed that estradiol selectively up-regulates iNOS expression in the oviduct during the periovulatory period corresponding to the window of ovulation, oocyte transport, and fertilization. The resulting NO production by this high-output NOS may be of crucial importance for reproductive events that occur in the oviduct. This study provided the first demonstration that NO production is hormonally regulated in the mammalian oviducts in vivo. Our results suggest that neuronal NOS, iNOS, and endothelial NOS contribute to oviductal functions in a timely and site-specific manner.     

Endothelial,but not the inducible,nitric oxide synthase is detectable in normal and portal hypertensive rats

BACKGROUND: Chronic portal hypertension is accompanied by a nitric oxide (NO) dependent vasodilation. Three isoforms of NO producing synthases (NOS) are characterized: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Sources of increased NO levels in chronic hypertension is disputed. METHODS: To determine eNOS and iNOS expression in different organs of portal hypertensive and control rats, we divided Sprague-Dawley rats in 6 groups: (1). Partial portal vein ligated rats, (2). Bile duct ligated rats, (3). Carbon tetrachloride treated rats, (4). Sham operated rats, (5). Untreated control rats, and (6). LPS treated rats. Immunohistochemistry (IHC) and immunoblotting (IB) using antibodies against eNOS or iNOS were carried out on samples from thymus, aorta, heart, lung, oesophagus, liver, spleen, kidney, pancreas, small and large intestine. RESULTS: IHC revealed an even eNOS expression in all groups. Expression of iNOS was restricted to macrophages in organs of LPS treated and the thymus of rats. IB mirrored these results. CONCLUSION: In chronic portal hypertension, the main source for NO production depends on eNOS activity.     

Endothelial,but not the inducible,nitric oxide synthase is detectable in normal and portal hypertensive rats

Abstract: Background: Chronic portal hypertension is accompanied by a nitric oxide (NO) dependent vasodilation. Three isoforms of NO producing synthases (NOS) are characterized: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Sources of increased NO levels in chronic hypertension is disputed. Methods: To determine eNOS and iNOS expression in different organs of portal hypertensive and control rats, we divided Sprague-Dawley rats in 6 groups: (1) Partial portal vein ligated rats, (2) Bile duct ligated rats, (3) Carbon tetrachloride treated rats, (4) Sham operated rats, (5) Untreated control rats, and (6) LPS treated rats. Immunohistochemistry (IHC) and immunoblotting (IB) using antibodies against eNOS or iNOS were carried out on samples from thymus, aorta, heart, lung, oesophagus, liver, spleen, kidney, pancreas, small and large intestine. Results: IHC revealed an even eNOS expression in all groups. Expression of iNOS was restricted to macrophages in organs of LPS treated and the thymus of rats. IB mirrored these results. Conclusion: In chronic portal hypertension, the main source for NO production depends on eNOS activity.     

Expression of endothelial nitric oxide synthase and inducible nitric oxide synthase in synovium of rheumatoid arthritis]

OBJECTIVES: To examine the localization and distribution of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), which participate in nitric oxide (NO) production, in synovium of rheumatoid arthritis (RA). MATERIALS AND METHODS: Immunohistochemical analysis for eNOS and iNOS in synovial tissues obtained from 10 patients with RA who were underwent total knee replacement. Synovial tissues of osteoarthritis (OA) were used as control. The percentage of cells that were positive for eNOS and iNOS was estimated in five hundred endothelial cells, synovial lining cells and interstitial cells, respectively. And mRNA expression of NOS was confirmed by in situ hybridization. In addition, to test NO production, nitration of tyrosines was assessed by immunohistochemistry. RESULTS: Not only endothelial cells but also synovial lining cells and interstitial cells exhibited immune-reactive both eNOS and iNOS. Cells which were seemed immune-reactive eNOS and iNOS expressed nitrotyrosin. By in situ hybridization, we detected mRNA expression for eNOS and iNOS. CONCLUSIONS: Endothelial cells, synovial lining cells and interstitial cells expressed both eNOS and iNOS with high frequency in RA synovium compared with OA synovium. It seemed to correlate with NO production. These results suggest that expression of iNOS may be involved in the induction of arthritis and eNOS may be participated in augmentation of inflammation in RA.     

Cystic fibrosis lung disease: the role of nitric oxide

This review summarizes current knowledge about the role of nitric oxide (NO) in cystic fibrosis (CF) lung disease. NO is endogenously produced by a group of enzymes, the NO synthases (NOSs). There are three isoforms of NOS, each encoded by different genes: neuronal (nNOS), immune or inducible (iNOS), and endothelial (eNOS) nitric oxide synthase.(1) They all form NO and L-citrulline by enzymatic oxidation of L-arginine. This reaction requires a number of cosubstrates, including molecular oxygen and tetrahydrobiopterin. It is now known whether all three isoenzymes are constitutively expressed in cells of the respiratory tract and that their gene expression is inducible.(2,3) NO production by iNOS, the "high-output" NOS, is stimulated by bacterial lipopolysaccharide (LPS) as well as proinflammatory cytokines such as interleukin (IL)-1gamma, IL-2, interferon (IFN)-gamma, and tumor necrosis factor (TNF). In contrast to nNOS and eNOS, activation of iNOS does not require an increase in intracellular Ca(2+) concentration.     

Expression and relationship between endothelin-1 messenger ribonucleic acid (mRNA) and inducible/endothelial nitric oxide synthase mRNA isoforms from normal and preeclamptic placentas

Preeclampsia is a mainly vascular disease of pregnancy, probably caused by an imbalance between vasodilator and vasoconstrictor agents that results in generalized vasospasm and poor perfusion in many organs. Among these factors, endothelin-1 (ET-1), a potent vasoconstrictor, is highly increased in preeclamptic women, while nitric oxide (NO), a vasodilator of human utero-placental arteries, is reduced in the same patients. The present study was designed to investigate the interactions between ET-1 and the NO system in the feto-placental unit; to this purpose we also examined the messenger ribonucleic acid (mRNA) expression of ET-1, inducible NO synthase (iNOS), and endothelial NOS (eNOS) in human cultured placental trophoblastic cells obtained from preeclamptic (PE) and normotensive (NT) pregnancies. We also studied whether exogenous ET-1 may affect the expression of iNOS and eNOS in human placental trophoblastic cells. Interestingly, by Northern blot analysis we observed an increased ET-1 mRNA expression level in PE trophoblastic cells compared to NT trophoblastic cells. Furthermore, exogenous ET-1 (10(-7) mol/L) was able to up-regulate its own mRNA expression in both NT and PE trophoblastic cells. iNOS and eNOS mRNA expression was then detected, by semiquantitative PCR, in both NT and PE trophoblastic cells. PE trophoblastic cells expressed lower iNOS mRNA levels compared with NT pregnancies. On the contrary, eNOS mRNA expression was higher in PE trophoblastic cells than in NT cells. Moreover, in the presence of ET-1 we observed a decrease in iNOS and an increase in eNOS mRNA expression levels in both NT and PE trophoblastic cells compared with the respective untreated cells. In conclusion, we demonstrate that ET-1 expression is increased in PE cells, whereas iNOS, which represents the main source of NO synthesis, is decreased; conversely, eNOS expression is increased. Finally, ET-1 is able to influence its own as well as NOS isoform expression in normal and PE trophoblastic cultured cells. These findings suggest the existence of a functional relationships between ET(s) and NOS isoforms that could constitute the biological mechanism leading to the reduced placental blood flow and increased resistance to flow in the feto-maternal circulation, which are characteristic of the pathophysiology of preeclampsia.     

Endometrial and myometrial expression of nitric oxide synthase isoforms in pre- and postmenopausal women.

Expression of nitric oxide synthase (NOS) protein was examined by Western immunoblot analysis and immunohistochemistry in the endometrium and myometrium of 19 premenopausal and 18 postmenopausal women undergoing hysterectomy for benign gynecological reasons. The predominant isoform of NOS in the human uterus was endothelial NOS (eNOS). Using immunohistochemistry, eNOS was localized predominantly to the glandular epithelium and endometrial microvascular endothelium. eNOS was scant and inconsistently detected in endometrial stromal cells. In the myometrium, eNOS was predominantly found in smooth muscle cells (myocytes) and was also detected in the microvascular endothelium. Neuronal NOS was not detectable by immunohistochemical techniques, and inducible NOS (iNOS) was only detectable in occasional specimens, although more often in secretory specimens. iNOS, when present, was predominantly found in glandular epithelium and occasional stromal cells. Myometrial iNOS was scant and not consistently detected. By Western immunoblot analysis, neuronal NOS or iNOS was not detected. We observed a unique menstrual cycle-dependent expression of eNOS that was different in the endometrium compared to the myometrium and was independent of uterine pathology. In the endometrium, there was 62% higher expression of eNOS during the secretory phase (P = 0.00085) compared to the proliferative phase, whereas in the myometrium, there was 74% greater expression of eNOS in the proliferative phase (P = 0.03) compared to the secretory phase. Within the secretory phase, maximal endometrial eNOS expression was found in the midportion, whereas in the myometrium, highest eNOS expression occurred during the late secretory phase. In postmenopausal women not treated with hormones, a significant reduction in endometrial and myometrial expression of eNOS occurred, which was reversed by continuous hormone replacement therapy. In summary, both endogenous ovarian steroids and exogenous sex hormones influence uterine eNOS expression. Our results suggest that estrogen may regulate myometrial eNOS, whereas progesterone or a combination of estrogen and progesterone may be more important in regulating endometrial eNOS, and NO may be a critical mediator of sex steroid actions in the human uterus.     

Expression patterns of endothelial and inducible nitric oxide synthase isoforms in corpora lutea of pseudopregnant rabbits at different luteal stages

Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P相似文献   

Inverse relationship between nitric oxide synthases and endothelin-1 synthesis in bovine corpus luteum: interactions at the level of luteal endothelial cell

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 F2alpha 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 F2alpha 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.     

Alterations in nitrate/nitrite and nitric oxide synthase in preovulatory follicles in gonadotropin-primed immature rat

Nitric oxide synthase (NOS) and nitric oxide (NO) play important roles in ovulation. The purpose of this study was to investigate the changes of intrafollicular nitrate/nitrite concentration and NOS mRNA expression in preovulatory follicles during equine CG (eCG) and human CG (hCG) induced ovulation in immature rats. Immature Sprague-Dawley rats received 15 IU eCG and then 15 IU hCG 48 h later. Rats were killed immediately before, 5 h after or 10 h after hCG injection, and their preovulatory follicles were dissected. Follicular fluid, granulosa cell, and theca cell layers were collected from preovulatory follicles and assayed for NO or NOS mRNA or for in vitro incubation study. Nitrate/nitrite concentration in the follicular fluid decreased significantly 5 and 10 h after hCG injection. Inducible NOS (iNOS) mRNA expression, which was greater in granulosa cell than in the theca cell layer, decreased significantly 5 and 10 h after hCG injection. However, endothelial NOS (eNOS) mRNA expression was detected mainly in the theca cell layer and further increased 5 and 10 h after hCG injection but remained low in granulosa cells. In vitro treatment of granulosa cells with 10(-4) or 5x10(-4) M S-nitroso-L-acetyl penicillamine (NO donor) decreased progesterone production and increased DNA fragmentation. We concluded that the decrease in nitrate/nitrite concentration in preovulatory follicles after hCG injection was due mainly to decreased iNOS expression in granulosa cells. These changes in nitrate/nitrite concentration may prevent apoptosis in preovulatory follicles.     

Distribution of constitutive nitric oxide synthase in the jejunum of adult rat

AIM：To study the distribution of the constitutive nitric oxide synthase(NOS) in the jejunom of adult rat.METHODS:The distribution of endothelial NOS(eNOS) was detected by immunohistochemistry.Immunofluorescence histochemical dual stainging technique were used for studying the distribution of neuronal NOS( nNOS) and eNOS,The dual stained slides were observed under a confocal laser scanning microscope.RESULTS:Positive neuronal NOS(nNOS) and endothelial NOS(eNOS) cells were found to be distributed in lamina propria of villi,and the epithelial cell was not stained,eNOS was mainly located in submucosal vascular endothelia while nNOS was mainly sityated in myenteric plexus.Some cells in the villi had both nNOS and eNOS.More than 80% of the cells were positive for both nNOS and eNOS,the rest cells were positive either for nNOS or for eNOS.CONCLUSION:The two constitutive nitric oxide synthases are distributed differently in the jejunum of rat.nNOS distributed in myenteric plexus is a neurotransmitter in the non-adrenergic non-cholinergic(NANC)inhibitory nerves eNOS distributed in endothelial and smooth muscle cells of blood vessels plays vasodilator role .eNOS and nNOS are coexpressed in some cells of lamina propria of villi.NO genearted y those NOS is very important in the physiological and pathological process of small intestine.     

Leptin receptor expression and in vitro leptin actions on prostaglandin release and nitric oxide synthase activity in the rabbit oviduct

In this study, we have examined the presence and the distribution of receptors for leptin (Ob-R) in the oviduct of rabbits, and the effects of leptin on the release of prostaglandin (PG) F2alpha and PGE2 and on the activity of nitric oxide (NO) synthase (NOS) by oviducts cultured in vitro. Rabbits were killed during the follicular phase and the oviducts were incubated in vitro with leptin, PGF2alpha, PGE2, NO donor and inhibitors of NOS and cyclo-oxigenase (COX). Using immunohistochemistry, Ob-R-like positive reaction was observed only in the cytoplasm of secretory cells, having stronger intensity in the infundibulum and ampulla tracts than in the isthmus. Both leptin and NO donor inhibited PGE2 release, whereas they enhanced PGF2alpha release; NOS inhibitor alone or with leptin increased PGE2 and decreased PGF2alpha production; NOS activity was enhanced by leptin, while PGs did not affect this enzyme. This study suggests that the oviduct could be a potential target for endocrine regulation by leptin, whose circulating levels may act as a metabolic signal modulating oviductal PG release through mediation of the NOS/NO system.     

Tumor necrosis factor-alpha-induced nitric oxide restrains the apoptotic response of anterior pituitary cells

We previously reported that tumor necrosis factor-alpha (TNF-alpha) inhibits cell proliferation whereas it stimulates apoptosis of anterior pituitary cells in an estrogen-dependent manner. Also, we showed that nitric oxide (NO) mediates the inhibitory effect of TNF-alpha on prolactin release. Here, we studied the effect of TNF-alpha on nitric oxide synthase (NOS) activity and expression in anterior pituitary cells from cycling and ovariectomized (OVX) rats, and the role of NO in TNF-alpha induced apoptosis of anterior pituitary cells. NOS activity was higher in anterior pituitary cells from rats in proestrus than in diestrus and was stimulated by 17beta-estradiol (10(-9) M, E2). TNF-alpha (50 ng/ml) stimulated NOS activity in anterior pituitary cells from rats at both stages of the estrous cycle and in cells from OVX rats cultured either with or without E2. Inducible NOS (iNOS) gene expression was higher in anterior pituitary cells from rats in proestrus than in diestrus and its expression was enhanced by TNF-alpha. Acute administration of E2 to OVX rats increased endothelial NOS (eNOS) expression in the anterior pituitary gland. Also, E2 increased eNOS mRNA in dispersed anterior pituitary cells from OVX rats, and this effect was blocked by TNF-alpha. nNOS expression in the anterior pituitary gland was higher at proestrus than at diestrus but eNOS expression was similar at both stages. TNF-alpha decreased eNOS mRNA in anterior pituitary cells from rats at proestrus or diestrus. In anterior pituitary cells from OVX rats, TNF-alpha failed to induce apoptosis but was able to induce it when cells were incubated with NAME or NMMA, NOS inhibitors that did not affect cell viability per se. In the presence of E2, NAME induced apoptosis and enhanced the proapoptotic effect of TNF-alpha. In conclusion, our study shows that TNF-alpha upregulates iNOS gene expression whereas it downregulates estrogen-induced eNOS expression in anterior pituitary cells. Endogenous NO may restrain rather than mediate the proapoptotic effect of TNF-alpha in anterior pituitary cells.     

Expression of nitric oxide synthase in human gastric carcinoma and its relation to p53, PCNA

AIM: To investigate the expression of NOS in gastric carcinoma, and to explore the relationship between the expression of nitric oxide synthases (NOS) and p53, PCNA, pathological features and clinical staging of gastric cancer. METHODS: The activity of NOS protein was investigated in 85 samples of human gastric carcinoma and 25 samples of normal gastric mucosal tissue by biochemical assay. We then examined the expression of NOS, p53, PCNA in 85 samples of human gastric cancer was examined by immunohistochemistry, and NOS mRNA expression in 85 gastric cancer tissue specimens by In situ hybridization. RESULTS: Biochemical assay showed that the activity of NOS was significantly higher in gastric carcinoma than in normal gastric mucosal tissues (t=0.4161, P＜0.01). Immunohistochemistry revealed that endothelial nitric oxide synthase (eNOS) expressed in all samples of normal gastric mucosa, but only 6 cases of 85 gastric cancer specimens showed weak positive immunohistochemical reactions to eNOS (20%). Inducible nitric oxide synthase (iNOS) was expressed strongly in human gastric carcinoma (81.2%). In situ hybridization analysis showed that iNOS mRNA expression was significantly stronger than eNOS mRNA expression in gastric cancer tissue (X~2 = 10.23, P＜0.01). The expression of iNOS in gastric cancer was associated with differentiation, clinical stages or lymph node metastases (r=0.3426,P＜0.05). However, iNOS expression did not correlate with histological classifications and morphological types. The expression of iNOS was significantly correlated with p53 or PCNA expression (r=0.3612, P＜0.05). The expression of neuronal nitric oxide synthase (nNOS) was not examined by immunohistochemistry and in situ hybridization in gastric cancer specimens and normal gastric mucosa. CONCLUSION: In human gastric cancer, there is an enhanced expression of iNOS, but not of eNOS. NOS promotes the proliferation of tumor cells and plays an important role in gastric cancer spread. Inactivation of antioncogene p53 and overexpression of iNOS might play a synergetic role in the process of carcinogenesis of human gastric carcinoma.     

Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo.

Nitric oxide (NO) is an important mediator of inflammatory responses in the lung and a key regulator of bronchomotor tone. An airway NO synthase (NOS; EC 1.14.13.39) has been proposed as a source of endogenous NO in the lung but has not been clearly defined. Through molecular cloning, we conclusively demonstrate that NO synthesis in normal human airways is due to the continuous expression of the inducible NOS (iNOS) isoform in airway epithelial cells. Although iNOS mRNA expression is abundant in airway epithelial cells, expression is not detected in other pulmonary cell types, indicating that airway epithelial cells are unique in the continuous pattern of iNOS expression in the lung. In situ analysis reveals all airway epithelial cell types express iNOS. However, removal of epithelial cells from the in vivo airway environment leads to rapid loss of iNOS expression, which suggests expression is dependent upon conditions and/or factors present in the airway. Quantitation of NOS activity in epithelial cell lysates indicates nanomolar levels of NO synthesis occur in vivo. Remarkably, the high-level iNOS expression is constant in airway epithelium of normal individuals over time. However, expression is strikingly decreased by inhaled corticosteroids and beta-adrenergic agonists, medications commonly used in treatment of inflammatory airway diseases. Based upon these findings, we propose that respiratory epithelial cells are key inflammatory cells in the airway, functioning in host defense and potentially playing a role in airway inflammation.     

The nitric oxide system response to hypoxia/reoxygenation in the aged cerebral cortex

Aged individuals are more susceptible to hypoxic insults, but little is known about the response of the nitric oxide (NO) system to hypoxia in the senescent brain. We have analysed the effect of aging on the hypobaric hypoxia/reoxygenation NO synthase (NOS) expression and activity in the cerebral cortex. In aged animals, the absence of significant changes in NOx and activity indicates a weaker response of the systems involving NO production in this pathological situation. The nNOS protein levels remained invariable and similar in both age groups after hypoxia, although in aged animals the mRNA did not change and was consistently lower than in adults. Both eNOS mRNA and protein increased shortly after hypoxia. However, although eNOS protein levels were quite similar in both age groups, the increase appeared later and was less persistent in aged animals. Real-time RT-PCR revealed a similar basal inducible NOS (iNOS) mRNA expression that responded late in reoxygenation, mainly in aged rats. However, neither iNOS protein nor activity was detected in any age group. Altogether our results indicate that aging attenuates the response of the NO system to a hypoxic injury, particularly at eNOS level, the activity of which is crucial for maintaining vascular homeostasis.