Red blood cells express a functional endothelial nitric oxide synthase

The synthesis of nitric oxide (NO) in the circulation has been attributed exclusively to the vascular endothelium. Red blood cells (RBCs) have been demonstrated to carry a nonfunctional NO synthase (NOS) and, due to their huge hemoglobin content, have been assumed to metabolize large quantities of NO. More recently, however, RBCs have been identified to reversibly bind, transport, and release NO within the cardiovascular system. We now provide evidence that RBCs from humans express an active and functional endothelial-type NOS (eNOS), which is localized in the plasma membrane and the cytoplasm of RBCs. This NOS is regulated by its substrate L-arginine, by calcium, and by phosphorylation via PI3 kinase. RBC-NOS activity regulates deformability of RBC membrane and inhibits activation of platelets. The NOS-dependent conversion of L-arginine in RBCs is comparable to that of cultured human endothelial cells. RBCs in eNOS-/- mice in contrast to wild-type mice lack NOS protein and activity, strengthening the evidence of an eNOS in RBCs. These data show an eNOS-like protein and activity in RBCs serving regulatory functions in RBCs and platelets, which may stimulate new approaches in the treatment of NO deficiency states inherent to several vascular and hematologic diseases.     

Correction of endothelial dysfunction in diabetic female rats by tetrahydrobiopterin and chronic insulin

Diabetes-induced vascular dysfunction has mainly been studied in males. However, the mechanisms involved may not correspond to those in females. Here we analyzed the effects of tetrahydrobiopterin (BH(4)) and chronic insulin on the physiology of mesenteric arterioles of alloxan-diabetic female rats. The parameters studied were the mesenteric arteriolar reactivity (intravital microscopy), nitric oxide synthase (NOS) activity (conversion of L-arginine to L-citrulline), eNOS gene expression (RT-PCR), NO production (diaminofluorescein), reactive oxygen species (ROS) generation (intravital fluorescence microscopy) and Cu/Zn superoxide dismutase (SOD) activity (spectrophotometry) and gene expression (RT-PCR). The reduced endothelium-dependent vasodilation of diabetic females was corrected by both BH(4) and insulin. NOS activity was decreased by diabetes, but insulin did not correct it. However, NOS expression was not modified by either diabetes or insulin. Arterioles of diabetic rats exhibited lower NO production, which was fully corrected by BH(4) and only partially by insulin. ROS generation was increased in diabetic rats, and both BH(4) and insulin normalized it. Diabetes did not change SOD activity and gene expression. However, insulin increased SOD activity but not its expression. Our data suggest that, similarly to males, endothelial dysfunction in female diabetic rats involves an altered ROS/NO imbalance. In contrast to males, however, insulin does not regulate NOS in the microcirculation of diabetic females.     

Asymmetric dimethylarginine levels in thyroid diseases

Thyroid diseases may lead to endothelial dysfunction; however, the mechanism underlying the endothelial dysfunction in thyroid disease is not clear yet. Asymmetric dimethylarginine (ADMA), a novel inhibitor of endothelial nitric oxide synthase (eNOS), blocks nitric oxide (NO) synthesis from L-arginine. Symmetric dimethylarginine (SDMA) is the structural isomer of the eNOS inhibitor ADMA. SDMA does not directly inhibit eNOS but is a competitive inhibitor of arginine transport. Increased plasma ADMA, SDMA concentrations, and low L-arginine/ADMA ratio were considered as possible contributing factors for endothelial dysfunction in hyperthyroid patients. On the other hand, plasma ADMA, SDMA levels and L-arginine/ADMA ratio in the hypothyroid group were unexpectedly found to be similar to those of the control subjects. The aim of this study is to evaluate and compare the plasma ADMA levels in hyperthyroid, hypothyroid and healthy subjects. Plasma ADMA, SDMA, and L-arginine levels were measured by high performance liquid chromatography. Plasma ADMA levels were significantly higher in both patients with hyperthyroidism and hypothyroidism than in the control group. SDMA concentrations were significantly increased in hypothyroid patients compared to control subjects. Patients with hyperthyroidism and hypothyroidism had significantly higher plasma L-arginine levels compared with healthy controls. L-arginine/ADMA ratio, which shows NO bioavailability, was significantly lower in hyperthyroid patients than in both hypothyroid and control subjects. In hyperthyroidism, plasma ADMA levels were related to age, L-arginine, and SDMA levels. SDMA was associated with age and L-arginine. L-arginine/ADMA ratio was negatively associated with freeT4 levels. There was a relationship between ADMA and L-arginine in hypothyroid patients. SDMA was significantly related to L-arginine, total cholesterol, and LDL. In conclusion, not only hyperthyroidism but also hypothyroidism was associated with alterations of ADMA and SDMA metabolism.     

替罗非班对大鼠心肌缺血再灌注后无复流及一氧化氮合酶活性和一氧化氮含量的影响

目的研究替罗非班对大鼠心肌缺血再灌注后无复流及一氧化氮合酶(NOS)活性、一氧化氮(NO)含量的影响,探讨替罗非班改善心肌缺血再灌注后无复流的作用机制。方法雄性Wistar大鼠,随机分为假手术组、对照组和替罗非班组。建立急性心肌缺血再灌注无复流模型,用硫黄素S活体染色,观察大鼠心肌无复流范围;伊文斯蓝、氯化三苯基四氮唑(TTC)染色评估大鼠心肌缺血及梗死范围;紫外分光光度计测定缺血再灌注120min时缺血区心肌内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)、总一氧化氮合酶(tNOS)活性及NO含量。结果缺血再灌注后120min,替罗非班组大鼠与对照组大鼠心肌缺血范围相似[(43.13±5.69)%比(39.98±3.75)%,P>0.05],但无复流范围及梗死范围明显小于对照组[(34.36±6.04)%比(52.09±6.89)%,P<0.01;(80.41±8.48)%比(90.13±5.72)%,P<0.05);对照组大鼠心肌的eNOS活性低于假手术组,iNOS、tNOS活性及NO含量高于假手术组(P<0.01);替罗非班组大鼠心肌的iNOS活性及NO含量高于假手术组(P<0.05,P<0.01),eNOS、tNOS活性与假手术组差异无统计学意义。与对照组比较,替罗非班组大鼠心肌的eNOS活性高于对照组(P<0.05),iNOS活性及NO含量低于对照组(P<0.05,P<0.01),tNOS活性较对照组有降低的趋势,但差异无统计学意义。结论大鼠心肌缺血90min再灌注120min可发生无复流现象;替罗非班可缩小无复流及梗死范围,其机制可能与保护内皮功能有关。     

Stretch activates nitric oxide production in pulmonary vascular endothelial cells in situ

Whereas endothelial responses to shear stress have been studied extensively, the responses to circumferential vascular stretch are yet poorly defined. Circumferential stretch in pulmonary microvessels is largely determined by the transmural pressure gradient, hence by both vascular perfusion and alveolar ventilation pressures. Here, we have studied the production of nitric oxide (NO) by the endothelial nitric oxide synthase (eNOS) in two different models of vascular stretch in the intact lung: In isolated-perfused rat lungs, vascular stretch was induced by elevation of vascular pressure. In situ digital fluorescence microscopy revealed stretch-dependent NO production, which was localized to capillary endothelial cells and inhibited by NOS blockers. In isolated-perfused mouse lungs, vascular stretch was generated by ventilation with elevated negative pressure. Stretch-induced phosphorylation of Akt and eNOS in lung endothelial cells was demonstrated by immunohistochemistry and increased NO production by in situ fluorescence microscopy. Stretch-induced endothelial responses in both models were abrogated by pretreatment with phosphatidylinositol-3-OH kinase inhibitors. These findings demonstrate that circumferential stretch activates NO production in pulmonary endothelial cells by a signaling cascade involving phosphatidylinositol-3-OH kinase, Akt, and eNOS and that this response is independent from the mechanical factors causing vascular distension.     

Intrauterine growth retardation is associated with reduced activity and expression of the cationic amino acid transport systems y+/hCAT-1 and y+/hCAT-2B and lower activity of nitric oxide synthase in human umbilical vein endothelial cells

Intrauterine growth retardation (IUGR) is associated with vascular complications leading to hypoxia and abnormal fetal development. The effect of IUGR on L-arginine transport and nitric oxide (NO) synthesis was investigated in cultures of human umbilical vein endothelial cells (HUVECs). IUGR was associated with membrane depolarization and reduced L-arginine transport (V(max)= 5.8+/-0.2 versus 3.3+/-0.1 pmol/microg protein per minute), with no significant changes in transport affinity (K(m)=159+/-15 versus 137+/-14 micromol/L). L-Arginine transport was trans-stimulated (8- to 9-fold) in cells from normal and IUGR pregnancies. IUGR was associated with reduced production of L-[3H]citrulline from L-[3H] arginine, lower nitrite and intracellular L-arginine, L-citrulline, and cGMP. IUGR decreased hCAT-1 and hCAT-2B mRNA, and increased eNOS mRNA and protein levels. IUGR-associated inhibition of L-arginine transport and NO synthesis, and membrane depolarization were reversed by the NO donor S-nitroso-N-acetyl-L,D-penicillamine. In summary, endothelium from fetuses with IUGR exhibit altered L-arginine transport and NO synthesis (L-arginine/NO pathway), reduced expression and activity of hCAT-1 and hCAT-2B and reduced eNOS activity. Alterations in L-arginine/NO pathway could be critical for the physiological processes involved in the etiology of IUGR in human pregnancies.     

Endothelial arginase: A new target in atherosclerosis

Decreased endothelial nitric oxide (NO) bioavailability as it relates to endothelial dysfunction plays an important role in various cardiovascular disorders, including atherosclerosis. Recent research has provided evidence that endothelial dysfunction in atherosclerosis is not primarily caused by decreased endothelial NO synthase (eNOS) gene expression, but rather deregulation of eNOS enzymatic activity, which contributes to the increased oxidative stress in atherosclerosis. Among other mechanisms, the substrate L-arginine is an important limiting factor for NO production. Emerging evidence demonstrates that L-arginine is not only converted to NO via eNOS, but also metabolized to urea and L-ornithine via arginase in endothelial cells. Hence, arginase competes with eNOS for the substrate L-arginine, resulting in deceased NO production. There are an increasing number of studies showing that enhanced arginase gene expression and/or activity contribute to endothelial dysfunction in various cardiovascular disorders, including atherosclerosis. Thus, endothelial arginase may represent a new therapeutic target in atherosclerosis.     

前列腺素EI对人脐静脉内皮细胞中NO和eNOS的影响

目的探讨前列腺素EI(prostaglandin EI,PGEI)对内皮细胞一氧化氮(NO)表达和内皮型一氧化氮合酶(eNOS)活性的影响。方法以人脐静脉内皮细胞(HUVEC)为实验对象,检测不同浓度PGEI作用不同时间后,细胞培养上清液和细胞中NO水平的变化,以及细胞eNOS活性的改变。结果(1)随着PGEI浓度的升高,eNOS的活性和NO的含量均逐渐增加(P<0.05);(2)短时间PGEI的干预对eNOS和NO的影响均不明显,24h后细胞中eNOS活性明显升高(P<0.05),NO的含量自12h起随时间延长而增加(P<0.05);(3)用不同PGEI浓度预处理,使TNF-α对eNOS活动的抑制作用减弱。结论PGEI可能通过诱导eNOS的表达,促进NO的释放,且可以重新激活被TNF-α抑制的eNOS活性。     

内皮型一氧化氮合酶与2型糖尿病血尿酸水平

一氧化氮可通过抑制黄嘌呤氧化酶的活性而影响血尿酸水平。内皮型一氧化氮合酶可催化精氨酸合成一氧化氮,其活性降低可影响一氧化氮的生成。2型糖尿病患者高尿酸血症的患病率高达30%。遗传和环境因素所致内皮型一氧化氮合酶活性降低与2型糖尿病患者血尿酸水平升高密切相关。     

Endothelial cellular senescence is inhibited by nitric oxide: implications in atherosclerosis associated with menopause and diabetes

Senescence may contribute to the pathogenesis of atherosclerosis. Although the bioavailability of nitric oxide (NO) is limited in senescence, the effect of NO on senescence and its relationship to cardiovascular risk factors have not been investigated fully. We studied these factors by investigating senescence-associated beta-galactosidase (SA-beta-gal) and human telomerase activity in human umbilical venous endothelial cells (HUVECs). Treatment with NO donor (Z)-1-[2-(2-aminoethyl)-N-(2-aminoethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) and transfection with endothelial NO synthase (eNOS) into HUVECs each decreased the number of SA-beta-gal positive cells and increased telomerase activity. The NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) abolished the effect of eNOS transfection. The physiological concentration of 17beta-estradiol activated hTERT, decreased SA-beta-gal-positive cells, and caused cell proliferation. However, ICI 182780, an estrogen receptor-specific antagonist, and L-NAME each inhibited these effects. Finally, we investigated the effect of NO bioavailability on high glucose-promoted cellular senescence of HUVECs. Inhibition by eNOS transfection of this cellular senescence under high glucose conditions was less pronounced. Treatment with L-arginine or L-citrulline of eNOS-transfected cells partially inhibited, and combination of L-arginine and L-citrulline with antioxidants strongly prevented, high glucose-induced cellular senescence. These data demonstrate that NO can prevent endothelial senescence, thereby contributing to the anti-senile action of estrogen. The ingestion of NO-boosting substances, including L-arginine, L-citrulline, and antioxidants, can delay endothelial senescence under high glucose. We suggest that the delay in endothelial senescence through NO and/or eNOS activation may have clinical utility in the treatment of atherosclerosis in the elderly.     

Effect of a high salt diet on microvascular antioxidant enzymes

High dietary salt intake decreases the endothelium-dependent dilation of skeletal muscle arterioles by inhibiting local nitric oxide (NO) activity without changing vascular smooth muscle responsiveness to NO. Under these conditions, microvascular walls show evidence of oxidative stress, and scavengers of reactive oxygen species (ROS) abolish this oxidative stress and restore normal arteriolar responses to acetylcholine (ACh). We tested the hypothesis that the salt-dependent appearance of microvascular ROS, and accompanying reduction in endothelium-dependent dilation, is due to a decrease in antioxidant enzyme expression or activity. We studied spinotrapezius muscle microvessels in rats fed normal (NS) (0.45%) or high (HS) (7%) salt diets for 4-5 weeks. Western analysis of arteriolar and venular protein showed no difference between groups in the content of superoxide dismutase (Cu/Zn SOD), catalase, or glutathione peroxidase. The catalase inhibitor 3-amino-1,2,4-triazole (3AT) increased arteriolar and venular oxidant activity (assessed by tetranitroblue tetrazolium reduction) by the same amount in both groups, suggesting similar levels of catalase activity. 3AT did not affect arteriolar responses to ACh in either group. The Cu/Zn SOD inhibitor diethyldithiocarbamate increased arteriolar and venular oxidant activity to a lesser extent in HS rats, suggesting reduced Cu/Zn SOD activity in this group. Cu/Zn SOD inhibition decreased arteriolar responses to ACh only in NS rats. These findings suggest that endogenous Cu/Zn SOD preserves the endothelium-dependent control of arteriolar tone in NS rats, and that a reduction in Cu/Zn SOD activity contributes to the loss of arteriolar NO activity in HS rats.     

Upregulation of vascular arginase in hypertension decreases nitric oxide-mediated dilation of coronary arterioles

One characteristic of hypertension is a decreased endothelium-dependent nitric oxide (NO)-mediated vasodilation; however, the underlying mechanism is complex. In endothelial cells (ECs), L-arginine is the substrate for both NO synthase (NOS) and arginase. Because arginase has recently been shown to modulate NO-mediated dilation of coronary arterioles by reducing l-arginine availability, we hypothesized that upregulation of vascular arginase in hypertension contributes to decreased NO-mediated vasodilation. To test this hypothesis, hypertension (mean arterial blood pressure >150 mm Hg) was maintained for 8 weeks in pigs by aortic coarctation. Coronary arterioles from normotensive (NT) and hypertensive (HT) pigs were isolated and pressurized for in vitro study. NT vessels dilated dose-dependently to adenosine (partially mediated by endothelial release of NO) and sodium nitroprusside (endothelium-independent vasodilator). Conversely, HT vessels exhibited reduced dilation to adenosine but dilated normally to sodium nitroprusside. Adenosine-stimulated NO release was increased approximately 3-fold in NT vessels but was reduced in HT vessels. Moreover, arginase activity was 2-fold higher in HT vessels. Inhibition of arginase activity by N(omega)-hydroxy-nor-l-arginine or incubation with l-arginine partially restored NO release and dilation to adenosine in HT vessels. Immunohistochemistry showed that arginase expression was increased but NOS expression was decreased in arteriolar ECs of HT vessels. These results suggest that NO-mediated dilation of coronary arterioles is inhibited in hypertension by an increase in arginase activity in EC, which limits l-arginine availability to NOS for NO production. The inability of arginase blockade or l-arginine supplementation to completely restore vasodilation may be related to downregulation of endothelial NOS expression.     

糖基化终产物对人脐静脉内皮细胞一氧化氮合酶活性和表达的影响及二甲双胍的保护作用

目的 探讨糖基化终产物及二甲双胍对人脐静脉内皮细胞一氧化氮合酶活性和表达的影响.方法 用胶原酶法分离人脐静脉内皮细胞并加以培养.将内皮细胞与不同浓度的糖基化终产物和二甲双胍分别孵育3、6、12、24 h,CCK-8法测定人脐静脉内皮细胞增殖活性.硝酸还原酶法测定一氧化氮含量,分光光度法测定一氧化氮合酶活性,蛋白免疫印迹法检测内皮型一氧化氮合酶蛋白表达水平.结果 糖基化终产物抑制人脐静脉内皮细胞增殖,二甲双胍促进人脐静脉内皮细胞增殖.糖基化终产物抑制人脐静脉内皮细胞的一氧化氮生成和一氧化氮合酶活性(P<0.01),呈剂量、时间依赖关系.二甲双胍(与对照组相比)或与糖基化终产物共同干预(与糖基化终产物组相比)均增加人脐静脉内皮细胞一氧化氮生成和一氧化氮合酶活性(P<0.01).糖基化终产物与人脐静脉内皮细胞共同孵育24 h后,内皮型一氧化氮合酶表达水平明显下降;二甲双胍上调内皮型一氧化氮合酶的表达;与糖基化终产物组相比,糖基化终产物与二甲双胍共同干预组内皮型一氧化氮合酶表达上调(P<0.01).结论 二甲双胍能够改善糖基化终产物导致的人脐静脉内皮细胞损伤.     

Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability

The molecular mechanisms of endothelial nitric oxide synthase (eNOS) regulation of microvascular permeability remain unresolved. Agonist-induced internalization may have a role in this process. We demonstrate here that internalization of eNOS is required to deliver NO to subcellular locations to increase endothelial monolayer permeability to macromolecules. Using dominant-negative mutants of dynamin-2 (dyn2K44A) and caveolin-1 (cav1Y14F), we show that anchoring eNOS-containing caveolae to plasma membrane inhibits hyperpermeability induced by platelet-activating factor (PAF), VEGF in ECV-CD8eNOSGFP (ECV-304 transfected cells) and postcapillary venular endothelial cells (CVEC). We also observed that anchoring caveolar eNOS to the plasma membrane uncouples eNOS phosphorylation at Ser-1177 from NO production. This dissociation occurred in a mutant- and cell-dependent way. PAF induced Ser-1177-eNOS phosphorylation in ECV-CD8eNOSGFP and CVEC transfected with dyn2K44A, but it dephosphorylated eNOS at Ser-1177 in CVEC transfected with cav1Y14F. Interestingly, dyn2K44A eliminated NO production, whereas cav1Y14F caused reduction in NO production in CVEC. NO production by cav1Y14F-transfected CVEC occurred in caveolae bound to the plasma membrane, and was ineffective in causing an increase in permeability. Our study demonstrates that eNOS internalization is required for agonist-induced hyperpermeability, and suggests that a mechanism by which eNOS is activated by phosphorylation at the plasma membrane and its endocytosis is required to deliver NO to subcellular targets to cause hyperpermeability.     

Nitric oxide and intestinal inflammation

Inflammation of the intestinal tract remains a very serious concern in the clinical setting. Unfortunately, to date, the mechanisms underlying many inflammatory conditions such as sepsis or inflammatory bowel diseases are poorly understood and our therapeutic interventions are less than ideal. Over the past decade, an abundance of research has been directed toward the role of nitric oxide (NO) in intestinal inflammation. It has become apparent that NO might have a dichotomous role as both a beneficial and detrimental molecule. Nitric oxide is a weak radical produced from L-arginine via the enzyme nitric oxide synthase (NOS). NOS exists in three distinct isoforms; constitutively (cNOS) expressed neuronal NOS (NOS1 or nNOS) and endothelial NOS (NOS3 or eNOS) or an inducible isoform (NOS2 or iNOS) capable of high production output of NO during inflammation. Constitutively expressed NOS has been shown to be critical to normal physiology and inhibition of these enzymes (nNOS or eNOS) caused damage. It has been proposed that the high output production of NO from iNOS causes injury, perhaps through the generation of potent radicals such as peroxynitrite and hence may explain the apparent dichotomous role of NO. However, recent studies have challenged this simple paradigm providing evidence that iNOS may have some protective role in some inflammatory models. Moreover, the importance of peroxynitrite has been questioned. In this review we discuss the role of cNOS and iNOS in intestinal inflammation and provide an overview of peroxynitrite in intestinal inflammation, highlighting some of the controversy that exists.     

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.     

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.     

Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells

Despite originally identified in neurones, the neuronal type of nitric oxide synthase (nNOS) is present also in cardiac and skeletal myocytes. Whether nNOS is functionally expressed in human endothelial cells--as the endothelial enzyme (eNOS)--is unknown. Human umbilical vein endothelial cells (HUVEC) were studied under control culture conditions and after 48 h treatment with cytomix (human tumour necrosis factor-alpha, interferon-gamma and E. coli endotoxin). We tested: (i) localisation and expression of nNOS and eNOS proteins by immunostaining and immunoblotting; (ii) activity of nNOS and eNOS by measuring L-arginine to L-citrulline conversion with 1-(2-trifluoromethylphenyl)imidazole (TRIM), a specific nNOS antagonist, in sub-cellular fractions; (iii) intracellular cGMP levels, as a marker for nitric oxide production, after TRIM pre-treatment, by radioimmunoassay. nNOS protein was expressed in the cytosolic fraction and immunolocalised in cultured HUVEC, and co-localised with the eNOS protein in frozen sections of the human umbilical cord. nNOS protein contributed to total L-citrulline production as TRIM selectively and dose-dependently reduced L-citrulline synthesis in the cytosolic but not particulate fraction of HUVEC. Similarly, TRIM reduced intracellular cGMP content both at baseline and after stimulation with a calcium ionophore. Cytomix down-regulated the expression and function of both nNOS and eNOS while no inducible NOS (iNOS) was detected. In conclusion, a functional neuronal type of NOS is co-expressed with the endothelial NOS type in HUVEC, suggesting a possible role for nNOS in regulation of blood flow.