Enhanced Nitric Oxide Synthesis Research Articles

Mixture of MMP-2, MLC, and NOS Inhibitors Affects NO Metabolism and Protects Heart from Cardiac I/R Injury.

Objectives Coronary reperfusion procedure leads to ischemia/reperfusion injury of the heart (IRI). IRI arises from increased degradation of myosin light chains and increased activity of matrix metalloproteinase 2 (MMP-2). Increased production of toxic peroxynitrite (ONOO−) during oxidative stress is a source of increased nitration/nitrosylation of contractile proteins, which enhance their degradation through MMP-2. Hence, an imbalance in nitric oxide (NO) metabolism along with oxidative stress is an important factor contributing to pathophysiology of cardiovascular disorders, including myocardial infarction. The aim of the current study was to provide an important insight into understanding the interaction of iNOS, eNOS, and ADMA during oxidative stress and to propose the beneficial therapy to modulate this interaction. Material and Methods. Pathogen-free Wistar rats were used in this study as a surrogate heart model ex vivo. Rat hearts perfused using the Langendorff method were subjected to global no-flow ischemia with or without administration of DOXY (1 µM), ML-7 (0.5 µM), and L-NAME (2 µM) mixture. Haemodynamic parameters of heart function, markers of I/R injury, tissue expression of iNOS, eNOS, and phospho-eNOS, asymmetric dimethylarginine, and NO production as well as MMP-2 activity were measured. Results Mechanical heart function and coronary flow (CF) were decreased in the hearts subjected to I/R. Treatment of the hearts with the tested mixture resulted in a recovery of mechanical function due to decreased activity of MMP‐2. An infusion of Doxy, ML-7, and L-NAME mixture into I/R hearts decreased the expression of iNOS, eNOS, and phospho-eNOS and in consequence reduced ADMA expression. Decreased ADMA production led to enhanced NO synthesis and improvement of cardiac function at 85% of aerobic control. Conclusions Synergistic effect of the multidrug therapy with the subthreshold doses allows addressing a few pathways of I/R injury simultaneously to achieve protection of cardiac function during I/R.

Hemoglobin-based Oxygen Carriers for Reversing Hypotension and Shock

Hemoglobin-based Oxygen Carriers for Reversing Hypotension and Shock

Endothelial regulation of eNOS, PAI-1 and t-PA by testosterone and dihydrotestosterone in vitro and in vivo

The aim of this study is the identification of direct endothelial regulation by the androgens testosterone (T) and dihydrotestosterone (DHT). We tested the effects of T and DHT on nitric oxide (NO) synthesis and on tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) expression in human endothelial cells and in ovariectomized (OVX) rats. The results showed that at physiological concentrations T and DHT increase endothelial synthesis of NO. This depends on a rapid recruitment of the extracellular-related kinase (ERK) 1/2 and of the phosphatidylinositol 3-OH kinase (PI3K)/Akt cascades, resulting in endothelial nitric oxide synthase (eNOS) Ser(1177)-phosphorylation. In addition, a later increase of eNOS expression is found. With supra-physiological amounts of T or DHT the induction of NO synthesis is lost. A concentration-related increase of t-PA expression starting from physiological concentrations of T or DHT is found, whereas PAI-1 is augmented only with higher doses. Although DHT exerts these actions through androgen receptors (AR), T acts in part through aromatase-dependent conversion to 17β-estradiol. Ovariectomy is associated with significant changes in eNOS, t-PA and PAI-1 expression in the aorta of Wistar rats and T and DHT result in modifications on eNOS, PAI-1 and t-PA that are in line with the in vitro experiments. In conclusion, T and DHT act on endothelial cells through AR or via conversion to estradiol. Physiological, but not higher amounts are associated with enhanced NO synthesis and an increased t-PA/PAI-1 ratio. These findings are useful to understand the impact of androgens in ageing individuals.

Genistein stimulates nitric oxide synthesis in human vascular endothelial cells via activation of cAMP‐response element binding protein

We previously reported that genistein, a phytoestrogen, up‐regulates endothelial nitric oxide synthase (eNOS) expression nitric oxide (NO) release in human vascular endothelial cells (ECs), an effect that is not related to its potential estrogenic action. In the present study, we further investigated the underlying mechanism by which genistein stimulates eNOS expression in ECs. Inhibition of the mitogen‐activated protein kinase, phosphatidylinositol 3‐OH kinase/Akt or protein kinase C had no effect on the enhanced NO synthesis by genistein. However, pharmacological or molecular intervention of PKA activation completely abolished PKA activity and the genistein‐stimulated eNOS expression and NO production, suggesting that genistein acts through a PKA‐dependent pathway. Furthermore, genistein increased intracellular cAMP concentration and PKA activity and subsequently activated the phosphorylation of CREB at ser‐133. Transfection of ECs with siRNA targeting CREB abolished genistein‐induced phosphorylation of CREB, eNOS expression and NO synthesis. These findings reveal for the first time an important signaling pathway in vascular ECs that is activated by genistein and suggest that this plant‐derived compound may play a beneficial role in vascular function through targeting cAMP/PKA/CREB/eNOS/NO signaling. This work was supported by grants from NCCAM/NIH (1R21AT004694‐01, 1R21AT002739).

Phospholipase D2 acts as an important regulator in LPS-induced nitric oxide synthesis in Raw 264.7 cells

The purpose of this study was to identify the role of phospholipase D2 (PLD2) in lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis. LPS enhanced NO synthesis and inducible nitric oxide synthase (iNOS) expression in macrophage cell line, Raw 264.7 cells. When Raw 264.7 cells were stimulated with LPS, the expressions of PLDs were increased. Thus, to investigate the role of PLD in NO synthesis, we transfected PLD1, PLD2, and their dominant negative forms to Raw 264.7 cells, respectively. Interestingly, only PLD2 overexpression, but not that of PLD1, increased NO synthesis and iNOS expression. Moreover, LPS-induced NO synthesis and iNOS expression were blocked by PLD2 siRNA, suggesting that LPS upregulates NO synthesis through PLD2. Next, we investigated the S6K1-p42/44 MAPK-STAT3 signaling pathway in LPS-induced NO synthesis mechanism. Knockdown of PLD2 with siRNA also decreased phosphorylation of S6K1, p42/44 MAPK and STAT3 induced by LPS. Furthermore, we found that STAT3 bound with the iNOS promoter, and their binding was mediated by PLD2. Taken together, our results demonstrate the importance of PLD2 for LPS-induced NO synthesis in Raw 264.7 cells with involvement of the S6K1-p42/44 MAPK-STAT3 pathway.

Involvement of nitric oxide in water stress-induced responses of cucumber roots

The effect of water deficit on nitric oxide (NO) generation was investigated in cucumber ( Cucumis sativus cv. Dar) seedling roots using bio-imaging with an NO-selective fluorophor, diaminofluorescein-2-diacetate (DAF-2DA). Roots subjected to mild (5 and 10 h) water deficit showed slightly enhanced NO synthesis in cells of root tips and in the surrounding elongation zone. However, severe (17 h) stress resulted in an intensive NO production localized mainly in and above the elongation zone. Water stress-induced NO generation was blocked by a specific NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) as well as nitrate reductase (NR) and partially by nitric oxide synthase (NOS-like) inhibitors. A pharmacological approach was used in order to verify the capacity of NO to induce adaptive responses of cucumber roots to water deficit. A positive correlation was found between NO donor (SNP 100 μM and GSNO 100 μM) pretreatment and plant hydration status, measured as relative water content (RWC) during progressive dehydration. At an early stage (5 h) of stress duration NO caused a periodical increase in lipoxygenase (LOX) activity, correlated with time-dependent enhancement of lipid peroxidation. Beginning from 10 h up to severe stress (17 h) exogenous NO was able to diminish LOX activity and alleviate water deficit-induced membrane permeability and lipid peroxidation, measured as TBARS content and visualised by histochemical staining in situ. Observed changes via NO were accompanied by a significant reduction of proline level, suggesting that the accumulation of this osmolyte might not be essential in water stress tolerance. Obtained results clearly indicate that NO augmentation is likely to help the plant at the initial stage of tissue dehydration to trigger efficient mechanisms, mitigating severe water deficit effects in roots of cucumber seedlings.

Nitric Oxide Synthesis is Modulated by 1,25-Dihydroxyvitamin D3 and Interferon-γ in Human Macrophages after Mycobacterial Infection

BackgroundLittle information is available the role of Nitric Oxide (NO) in host defenses during human tuberculosis (TB) infection. We investigated the modulating factor(s) affecting NO synthase (iNOS) induction in human macrophages.MethodsBoth iNOS mRNA and protein that regulate the growth of mycobacteria were determined using reverase transcriptase-polymerase chain reaction and western blot analysis. The upstream signaling pathways were further investigated using iNOS specific inhibitors.ResultsHere we show that combined treatment with 1,25-dihydroxyvitamin D3 (1,25-D3) and Interferon (IFN)-γ synergistically enhanced NO synthesis and iNOS expression induced by Mycobacterium tuberculosis (MTB) or by its purified protein derivatives in human monocyte-derived macrophages. Both the nuclear factor-κB and MEK1-ERK1/2 pathways were indispensable in the induction of iNOS expression, as shown in toll like receptor 2 stimulation. Further, the combined treatment with 1,25-D3 and IFN-γ was more potent than either agent alone in the inhibition of intracellular MTB growth. Notably, this enhanced effect was not explained by increased expression of cathelicidin, a known antimycobacterial effector of 1,25-D3.ConclusionThese data support a key role of NO in host defenses against TB and identify novel modulating factors for iNOS induction in human macrophages.

Inhibition of lipopolysaccharide-induced nitric oxide synthesis by nicotine through S6K1-p42/44 MAPK pathway and STAT3 (Ser 727) phosphorylation in Raw 264.7 cells

Lipopolysaccharide (LPS) has been known to produce inflammatory modulators such as tumor necrosis factor α (TNF-α) or nitric oxide (NO). In this study, we examined the effects of nicotine on LPS enhanced NO synthesis and inducible nitric oxide synthase (iNOS) expression in macrophages. LPS-induced NO synthesis and iNOS expression were significantly decreased by nicotine. To investigate the signaling mechanism of nicotine induced suppression of NO synthesis and iNOS expression induced by LPS, we focused on the possible roles of p42/44 MAPK, S6K1, and signal transducers and activators of transcription 3 (STAT3) signaling. LPS is known to activate p42/44 MAPK and S6K1, which in turn activates STAT3 to induce inflammatory regulators. Pretreatment of cells with nicotine blocked LPS-induced p42/44 MAPK and S6K1 as well as iNOS promoter activity. Furthermore, we found that LPS-induced phosphorylation of STAT3 at serine 727 is mediated by S6K1-p42/44 MAPK pathway, and this STAT3 phosphorylation was also blocked by nicotine. We also found that downregulation of STAT3 using STAT3 siRNA resulted in suppression of the NO synthesis and iNOS expression. Taken together, our results suggest that nicotine inhibits LPS-induced NO synthesis through suppression of S6K1-p42/44 MAPK pathway and phosphorylation of STAT3 in Raw 264.7 cells.

Genistein, a Soy Phytoestrogen, Upregulates the Expression of Human Endothelial Nitric Oxide Synthase and Lowers Blood Pressure in Spontaneously Hypertensive Rats1,

Genistein, a soy phytoestrogen, may improve vascular function, but the mechanism of this effect is unclear. Endothelial-derived nitric oxide (NO) is a key regulator of vascular tone and atherogenesis. Previous studies have established that estrogen can act directly on vascular endothelial cells (EC) to enhance NO synthesis through genomic stimulation of endothelial NO synthase (eNOS) expression. However, it is unknown whether genistein has a similar effect. We therefore investigated whether genistein directly regulates NO synthesis in primary human aortic EC (HAEC) and human umbilical vein EC (HUVEC). Genistein, at physiologically achievable concentrations in individuals consuming soy products, enhanced the expression of eNOS and subsequently elevated NO synthesis in both HAEC and HUVEC, with 1–10 μmol/L genistein inducing the maximal effects. However, the effects of genistein on eNOS and NO were not mediated by activation of estrogen signaling or inhibition of tyrosine kinases, 2 known biological actions of genistein. Genistein (1–10 μmol/L) increased eNOS gene expression (1.8- to 2.6-fold of control) and significantly increased eNOS promoter activity of the human eNOS gene in HAEC and HUVEC, suggesting that genistein activates eNOS transcription. Dietary supplementation of genistein to spontaneously hypertensive rats restored aortic eNOS levels, improved aortic wall thickness, and alleviated hypertension, confirming the biological relevance of the in vitro findings. Our data suggest that genistein has direct genomic effects on the vascular wall that are unrelated to its known actions, leading to increased eNOS expression and NO synthesis, thereby improving hypertension.

The application of sodium dextran sulfate to the field of cosmetics

In this study, it was shown that sodium dextran sulfate (DS), which is used as an antihyperlipoproteinemic and is clinically known for the improvement of the blood flow, accelerated nitric oxide (NO) synthesis in the normal human epidermal keratinocytes. Currently, the free radical NO is known to be an important mediator in various physiological and pathophysiological processes of the skin, such as regulation of blood flow. The result suggests that DS might exert improvement on the blood flow by the acceleration of NO synthesis. The evaluation in the human body test showed that DS effectively reduced and prevented the swelling of the lower leg. Consequently, it is reasonable to suggest that DS could penetrate into the epidermis and enhance NO synthesis, and NO released from the epidermal keratinocytes could promote the blood flow in the microvasculature and reduce the swelling of the lower leg.

Integrin alpha9beta1 promotes cell migration via Src kinase mediated nitric oxide synthesis

Beta-1 integrins are important mediators of cell migration but may do so through different signaling mechanisms. However, c-src-activation seems to be a key signaling intermediate. Nitric oxide (NO) synthesis is another novel molecule that modulates cell migration, which may be utilized during integrin mediated cell migration. We used α9 expressing SW480 colon carcinoma cells, to explore a role for src and NO in α9β1 mediated-cell migration. We found that α9β1-ligation leads to c-Src phosphorylation (Tyr-416) and Erk MAP kinase phosphorylation; phospho-c-src was maximum at 5 min, and phospho-Erk maxima was at 20min. Pre-treated cells with src and Erk kinase inhibitors, PP1 and PD98059, inhibited α9-dependent cell motility. c-src appeared to signal upstream of Erk since inhibition of c-Src-kinase blocked Erk phosphorylation. Furthermore, α9β1-ligation enhanced NO synthesis through activation of inducible Nitric Oxide Synthase (iNOS), which was constitutively expressed in SW480 cells. Using the NO specific inhibitor L-NAME, α9β1-dependent cell migration was blocked. Also, enhanced iNOS phosphorylation and NO production were src kinase (but not Erk) dependent. These results suggest a novel signaling pathway for α9β1-induced cell migration through c-src kinase activation of iNOS and subsequent NO production.

Protection of Endothelial Function: Targets for Nutritional and Pharmacological Interventions

The vascular endothelium synthesizes and releases a spectrum of vasoactive substances and therefore plays a fundamental role in the basal and dynamic regulation of the circulation. Nitric oxide (NO)-originally described as endothelium-derived relaxing factor-is released from endothelial cells in response to shear stress produced by blood flow, and in response to activation of a variety of receptors. After diffusion from endothelial to vascular smooth muscle cells, NO increases intracellular cyclic guanosine-monophosphate concentrations by activation of the enzyme guanylate cyclase leading to relaxation of the smooth muscle cells. NO has also antithrombogenic, antiproliferative, leukocyte-adhesion inhibiting effects, and influences myocardial contractility. Endothelium-derived NO-mediated vascular relaxation is impaired in spontaneously hypertensive animals. NO decomposition by free oxygen radicals is a major mechanism of impaired NO bioavailability. The resulting imbalance of endothelium-derived relaxing and contracting substances disturbs the normal function of the vascular endothelium. Endothelin acts as the natural counterpart to endothelium-derived NO. Besides its arterial blood pressure rising effect in humans, endothelin-1 induces vascular and myocardial hypertrophy, which are independent risk factors for cardiovascular morbidity and mortality. Current therapeutic strategies concentrate mainly on lowering low-density lipoprotein cholesterol and an impressive reduction in the risk for cardiovascular morbidity and mortality has been achieved. Inflammatory mechanisms play an important role in vascular disease and inflammatory plasma markers correlate with prognosis. The production of reactive oxygen species under pathological conditions may represent an important inflammatory trigger. Novel therapeutic strategies specifically targeting inflammation thus bear great potential for the prevention and treatment of atherosclerotic vascular disease. In this context, the vascular actions of flavanol-rich cocoa, particularly with regard to enhanced NO synthesis and endothelial function observed in humans following consumption, warrants further attention. This review discusses pharmacological and dietary intervention.

Tetrahydrobiopterin and Endothelial Dysfunction in Cardiovascular Diseases

Abstract Endothelial vasodilator dysfunction is a characteristic feature of patients at risk for coronary atheroscierosis. We have reported that insulin resistance may be a pathogenic factor for endothehal dysfunction through impaired endothelial nitric oxide synthase (eNOS) activity and increased oxidative breakdown of nitric oxide (NO) due to an enhanced fonnation of Superoxide anion, which arc caused by relative deficiency of tetrahydrobiopterin (BH4) in vascular endothelial cclls. Guanosine-triphosphate cyclohydrolase I, the rate-limiting cnzyme in the production of BH4, is decreased in the aorta of insulin-resistant rats and supplementation of BH4 restored the endothelial function and relieved oxidative tissue damage. The BH4 treatment may evoke these benefits not only by providing eNOS with cofactor to enhance NO synthesis, but also by acting as an indirect and/or direct antioxidant to decrease Superoxide anion derived from the endothelium. A further understanding of the physiological and pathological roles and their regulation may lead to new therapeutic avenues.

Dietary L-Arginine Supplementation Reduces Fat Mass in Zucker Diabetic Fatty Rats

This study was conducted to test the hypothesis that dietary supplementation of arginine, the physiologic precursor of nitric oxide (NO), reduces fat mass in the Zucker diabetic fatty (ZDF) rat, a genetically obese animal model of type-II diabetes mellitus. Male ZDF rats, 9 wk old, were pair-fed Purina 5008 diet and received drinking water containing arginine-HCl (1.51%) or alanine (2.55%, isonitrogenous control) for 10 wk. Serum concentrations of arginine and NO(x) (oxidation products of NO) were 261 and 70% higher, respectively, in arginine-supplemented rats than in control rats. The body weights of arginine-treated rats were 6, 10, and 16% lower at wk 4, 7, and 10 after the treatment initiation, respectively, compared with control rats. Arginine supplementation reduced the weight of abdominal (retroperitoneal) and epididymal adipose tissues (45 and 25%, respectively) as well as serum concentrations of glucose (25%), triglycerides (23%), FFA (27%), homocysteine (26%), dimethylarginines (18-21%), and leptin (32%). The arginine treatment enhanced NO production (71-85%), lipolysis (22-24%), and the oxidation of glucose (34-36%) and octanoate (40-43%) in abdominal and epididymal adipose tissues. Results of the microarray analysis indicated that arginine supplementation increased adipose tissue expression of key genes responsible for fatty acid and glucose oxidation: NO synthase-1 (145%), heme oxygenase-3 (789%), AMP-activated protein kinase (123%), and peroxisome proliferator-activated receptor gamma coactivator-1alpha (500%). The induction of these genes was verified by real-time RT-PCR analysis. In sum, arginine treatment may provide a potentially novel and useful means to enhance NO synthesis and reduce fat mass in obese subjects with type-II diabetes mellitus.

Nitric oxide involved in signal transduction of Jasmonic acid-induced stomatal closure ofVicia faba L.

Nitric oxide (NO) and Jasmonic acid (JA) are two key signaling molecules involved in many and diverse biological pathways in plants. Growing evidence suggested that NO signaling interacts with JA signaling. In this work, Our experiment showed that NO exists in guard cell ofVicia faba L., and NO is involved in signal transduction of JA-induced stomata closuring: (i) JA enhances NO synthesis in guard cell; (ii) both JA and NO induced stomatal closure, and had dose response to their effects; (iii) there are synergetic correlation between JA and lower NO concentration in regulation of stomatal movement; (iv) JA-induced stomatal closure was largely prevented by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger. An inhibitor of NO synthase (NOS) in mammalian cells,NG-nitro-L-Arg-methyl eater (L-NAME) also inhibits plant NOS, repressing JA-induced NO generation and JA-induced stomatal closure. We presumed that NO mainly comes from NOS after JA treatment.

17.BETA.-Estradiol Primes Elicitation of Inducible Nitric Oxide Synthase Expression by Lipopolysaccharide and Interferon-.GAMMA. in Mouse Macrophage Cell Line J774.1

The effects of estrogenic compounds on nitric oxide (NO) production by macrophages were examined. 17β-Estradiol promoted NO production triggered by lipopolysaccharide (LPS) and/or interferon (IFN)-γ in the mouse macrophage cell line J774.1. Other estrogen-like substances such as estrone, 17α-ethynylestradiol and bisphenol A also enhanced NO synthesis, but this NO synthesis was not activated by Ca2+ ionophore A23187. RT-PCR analysis demonstrated induction of inducible nitric oxide synthase (iNOS) mRNA in J774.1 cells exposed to 17β-estradiol. Although the estrogen receptor (ER)-antagonist ICI-182780 partially suppressed the promoting effect of 17β-estradiol on NOS activity, there was little ERα detectable by RT-PCR from J774.1 cells. These results suggest that ERs may participate only partially in iNOS mRNA transcription in J774.1 cells and that 17β-estradiol may act directly through other unknown intracellular signal transduction(s) that are activated by LPS and IFN-γ.

Enhancement of nitric oxide and proteoglycan synthesis due to cyclic tensile strain loaded on chondrocytes attached to fibronectin.

Mechanical stress is an essential factor in the pathogenesis of osteoarthrosis. We sought to determine whether the strain-mediated alteration in proteoglycan (PG) synthesis was modulated by nitric oxide (NO) synthesis. Cyclic tensile strain was applied to bovine articular chondrocytes. PG and NO synthesis were determined by [35S] sulfate incorporation and chemiluminescence analysis, respectively. To determine the expression of inducible NO synthase (iNOS), quantitative RT-PCR was used. Enhanced PG and NO synthesis were evident when cyclic tensile strain was applied to chondrocytes seeded on fibronectin-coated plates. When NO production was inhibited, PG synthesis was further enhanced. Cyclic tensile strain loaded on the chondrocytes enhanced NO synthesis and this enhanced NO inhibited PG synthesis.

Antagonistic effects of IL-4 and interferon-gamma (IFN-gamma) on inducible nitric oxide synthase expression in bovine macrophages exposed to gram-positive bacteria.

Cytokine-mediated modulation of nitric oxide (NO) production by bacteria-stimulated bovine macrophages was studied. When Salmonella dublin, as a prototypic gram-negative organism, was used, NO generation was barely enhanced by recombinant bovine and ovine IFN-gamma, but was suppressed by IL-4. Salmonella dublin-induced NO generation was not influenced by a panel of nine other cytokines. The panel included IL-1, tumour necrosis factor (TNF) and IFN-alpha, which are active in a similar mouse macrophage model. The tested cytokines were either homologous or known to interact with bovine cytokine receptors. Recombinant bovine and ovine IFN-gamma were the only cytokines which strongly enhanced NO synthesis by macrophages exposed to the gram-positive organism, Listeria monocytogenes. Listeria-induced NO generation was strongly suppressed by recombinant human and bovine IL-4, but not by IL-10 and transforming-growth-factor-beta. Thus, two cytokines characterizing a Th1 and a Th2 response up- and down-regulate, respectively, bacteria-induced NO generation in bovine macrophages, whereas nine other cytokines had little activity in this regard. This modulation was reflected in changes in the steady state levels of mRNA coding for inducible nitric oxide synthase. Combinations of IFN-gamma and IL-4 suggested that the relative proportion of these cytokines determined whether bacteria-induced NO generation was up- or down-regulated. At saturating IL-4 concentrations, stimulation of bacteria-induced NO generation in macrophages by T cell supernatants was solely dependent on IFN-gamma. This was shown by antibody neutralization experiments and by a close correlation between the capacity of supernatants to stimulate NO generation and the IFN-gamma content, as determined by immunoassay.

Enhanced nitric oxide activity offsets peripheral vasoconstriction during acute hypoxaemia via chemoreflex and adrenomedullary actions in the sheep fetus.

We tested the hypothesis that enhanced nitric oxide (NO) opposes fetal peripheral vasoconstrictor responses to acute hypoxaemia via actions involving the carotid chemoreflex and the adrenal medulla. The hypothesis was tested in the late gestation ovine fetus using a novel NO clamp technique, which involves fetal combined treatment with the NO synthase inhibitor, L-NAME, and the NO donor, sodium nitroprusside. In contrast to treatment with L-NAME alone, combined fetal treatment with L-NAME and nitroprusside prevents generalized vasoconstriction and pronounced hypertension, not only maintaining basal cardiovascular function, but also permitting blockade of the de novo synthesis of NO during hypoxaemia while compensating for the tonic production of the gas. Under general anaesthesia, seven sheep fetuses were surgically prepared with catheters and a femoral Transonic flow probe. Five days after surgery, fetuses were subjected to a 3 h protocol: 1 h normoxia, 1 h hypoxaemia and 1 h recovery. Fetal hypoxaemia was induced during either fetal infusion with saline or treatment with the NO clamp. During saline infusion, fetuses responded to hypoxaemia with transient bradycardia, femoral vasoconstriction and increases in plasma noradrenaline and adrenaline. During fetal treatment with the NO clamp, bradycardia persisted and there were greater peripheral vasoconstrictor and catecholaminergic responses to hypoxaemia. Further analysis showed that NO clamp treatment enhanced the chemoreflex component of the fetal cardiovascular defence to acute hypoxaemia. These data support the hypothesis that enhanced NO synthesis during acute hypoxaemia offsets fetal peripheral vasoconstrictor responses to hypoxaemia via chemoreflex and adrenomedullary actions.

Neuroinflammatory Role of Prostaglandins during Experimental Meningitis: Evidence Suggestive of an in Vivo Relationship between Nitric Oxide and Prostaglandins

Nitric oxide (NO) and prostaglandins are inflammatory mediators produced during meningitis. The purpose of the present study was to pharmacologically inhibit cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) to 1) explore the prostaglandin contribution to blood-cerebrospinal fluid barrier permeability alterations and 2) elucidate the in vivo concentration relationship between prostaglandin E2 (PGE2) and NO during experimental meningitis. Intracisternal injection of lipopolysaccharides (LPSs, 200 microg) induced neuroinflammation. Rats were dosed with nimesulide (COX-2 inhibitor), aminoguanidine (iNOS inhibitor), or vehicle. Evans blue was used to assess blood-cerebrospinal fluid barrier permeability. Meningeal NO and cerebrospinal fluid PGE2 were assayed using conventional methods. (Results are expressed as mean +/- S.E.M. of 5-9 rats/group.) Nimesulide failed to prevent blood-cerebrospinal fluid barrier disruption [cerebrospinal fluid Evans blue (micrograms per milliliter): control, 0.22 +/- 0.22*; LPS, 11.58 +/- 0.66; LPS + nimesulide, 10.58 +/- 0.86; *p < 0.05; ANOVA]. Although nimesulide decreased PGE2 (picograms per microliter; p < 0.01) in LPS + nimesulide rats (13.9 +/- 1.96) versus LPS + vehicle (73.8 +/- 12.4), meningeal NO production (picomoles/30 min/10(6) cells; p < 0.01) increased unexpectedly in LPS + nimesulide rats (439 +/- 47) versus LPS + vehicle rats (211 +/- 31). In contrast, aminoguanidine inhibited meningeal NO (picomoles/30 min/10(6) cells; p < 0.005) in LPS + aminoguanidine (111 +/- 20) versus LPS (337 +/- 48) but had no effects (p > 0.05) on PGE2. The in vivo relationship between PGE2 and NO was mathematically described by a biphasic, bell-shaped curve (r2 = 0.42; n = 27 rats; p < 0.0001). Based on these results, inhibition of prostaglandin synthesis not only fails to prevent blood-cerebrospinal fluid barrier disruption during neuroinflammation and but also promotes increased meningeal NO production. The in vivo concentration relationship between PGE2 and NO is biphasic, suggesting that inhibition of COX-2 alone may promote NO toxicity through enhanced NO synthesis.