Nitric Oxide Synthase eNOS Research Articles

English

The aim of this study was to investigate the efficiency of intrathecal glial cell line-derived neurotrophic factor (GDNF) on nitric oxide (NO) and nitric oxide synthase (NOS) activity following a spinal nerve ligation (SNL) of male Sprague Dawley rats. The rats were randomly divided into four groups: normal (control), sham-operated, SNL (SNL followed by a physiological saline injection into the subarachnoid space), and GDNF (SNL followed by a GDNF injection into the subarachnoid space). Each group was divided into three subgroups (n = 10). The rats in each subgroup were euthanised 3, 7, and 14 days after the operation. Rat behaviour was evaluated before euthanising, and the ipsilateral spinal cords were harvested after euthanising to determine the NO content and NOS activity. Compared with the control and sham-operated groups, the NO content and NOS activity in the SNL group increased significantly 3 days after the operation; this increase was maintained until 14 days after the operation (P < 0.01 or 0.05). A significant decrease was observed in the NO content and NOS activity in the GDNF group compared with the SNL group. The decrease continued until 14 days after the operation (P < 0.01 or 0.05). The results indicated that the NO and NOS activity in the rat spinal cord are associated with SNL-induced neuropathic pain. The decreased neuropathic pain from the intrathecal GDNF is correlated with the decrease in NO content and NOS activity in the spinal cord.   Key words: Glial cell line-derived neurotrophic factor, neuralgia, nitric oxide, nitric oxide synthase.

Glu298Asp polymorphism influences the effects of beetroot bread on diastolic blood pressure in healthy men: A pilot study

Glu298Asp polymorphism of the endothelial nitric oxide synthase eNOS gene (NOS3) has previously been identified as a risk factor for hypertension and coronary artery disease. Studies suggest that the higher risk observed in thymine (T) allele carriers is due to endothelial dysfunction associated with lower eNOS activity. It has been proposed that acute consumption of nitrate ameliorates postprandial endothelial dysfunction possibly by the donation of nitric oxide by a mechanism that does not rely on eNOS. Nevertheless, how these factors interact has yet to be determined. Therefore, the aim of this study was to investigate whether the presence of NOS3 Glu298Asp polymorphism interacts with the nitrate content of beetroot-enriched bread to influence postprandial blood pressure.

Orexin A regulates cardiovascular responses in stress-induced hypertensive rats

Several pieces of evidence indicate that the rostral ventrolateral medulla (RVLM) is probably one of the key neural structures mediating the pressor effects of orexins in the brain. Nitric oxide synthase/nitric oxide (NOS/NO) system in the RVLM modulates cardiovascular activities. Our experiments were designed to test the hypothesis that orexin-A (OXA) is involved in the mechanism of stress-induced hypertension (SIH) by adjusting NOS/NO system in the RVLM. The stress-induced hypertensive rats (SIHR) model was established by electric foot-shocks and noises. Here we examined the expression of OXA immunoreactive (OXA-IR) cells in the lateral hypothalamus (LH) and the protein level of orexin 1 receptor (OX1R) in the RVLM of SIHR, and we found that the expressions of OXA-IR and OX1R were higher than those of the control group. The double-staining immunohistochemical evidence showed that OX1R immunoreactive (OX1R-IR) cells and neuronal nitric oxide synthase (nNOS) or inducible nitric oxide synthase (iNOS) immunoreactive (IR) cells were co-localizated in the RVLM. Microinjection of OXA (10, 50, 100 pmol/100 nl) into the unilateral (right) RVLM of control rats or SIHR produced pressor and tachycardiac effects in a dose-dependent manner. SB-408124 (100 pmol/100 nl, an antagonist of OX1R) or TCS OX2 29 (100 pmol/100 nl, an antagonist of OX2R) partly abolished the cardiovascular effects of exogenously-administrated OXA into the RVLM of control rats and SIHR, and lowered the increased systolic blood pressure (SBP) and heart rate (HR) of SIHR, with no difference in statistical significance between the two antagonists' effects. Microinjection into the RVLM of both control and SIHR groups of 7-Ni (0.05 pmol/100 nl, nNOS inhibitor) or Methylene Blue [100 pmol/100 nl, an inhibitor of soluble guanylate cyclase (sGC)] suppressed the OXA-induced increase of SBP and HR, whereas microinjection of AG (1, 10, 100 pmol/100 nl) had no obvious effects on the OXA-induced increase of SBP and HR. Our results indicate that OXA in the RVLM may participate in the central regulation of cardiovascular activities in SIHR, and OX1R and OX2R both have important roles in it. The cardiovascular effects of OXA in the RVLM may be induced by nNOS-derived NO, which activated sGC-associated signaling pathway.

Defective Nitric Oxide–Cyclic Guanosine Monophosphate Signaling in Patients With Bipolar Disorder

Bipolar disorder (BD) is associated with elevated cardiovascular mortality rates. We investigated the modulation of l-arginine-nitric oxide (NO) signaling in platelets from patients with BD at different phases. Platelets obtained from 28 patients with BD and 10 healthy volunteers were analyzed for l-arginine transport, NO synthase (NOS) activity, cyclic guanosine monophosphate content, and biomarkers of oxidative stress. Expressions of NOS isoforms, soluble guanylyl cyclase, and arginase were also measured in platelets. Amino acid and C-reactive protein levels in plasma were assessed. Plasma concentrations of l-arginine (mean [M] ± standard error of the mean [SEM] = 97 ± 10 versus 121 ± 10 µM) and its transport into platelets (median [interquartile range] = 26.0 [28.6] versus 26.5 [43.9] pmol/10(9) cells per minute) did not differ between patients with BD and controls (p > .05). Patients with BD showed reduced NOS activity (M ± SEM = 0.037 ± 0.003 versus 0.135 ± 0.022 pmol/10(8) cells, p < .001), but not endothelial NOS, inducible NOS, and arginase expression, compared with controls (p > .05). Cyclic guanosine monophosphate content was reduced (M ± SEM = 0.022 ± 0.003 versus 0.086 ± 0.020 pmol/10(8) cells, p < .05) despite the absence of changes in soluble guanylyl cyclase expression (median [interquartile range] = 21.6 [15.5] versus 9.5 [9.4] arbitrary units, p > .05) in patients with BD. Superoxide dismutase activity, but not catalase activity, was increased in patients with BD in the manic phase (M ± SEM = 2094 ± 335 versus 172 ± 17 U/mg protein, p < .001). C-reactive protein was elevated only in manic episodes (M ± SEM = 0.8 ± 0.2 versus 0.1 ± 0.02 mg/L, p < .001). Impaired NO generation from platelets, inflammation, and oxidative stress may play pivotal roles in the multifaceted process of cardiovascular events in BD.

Abstract 239: Nitric Oxide Suppresses the Angiogeneic Potential of Mesenchymal Stem Cells

Introduction: Mesenchymal stem cells (MSCs) participate in blood vessel formation which is crucial for tissue regeneration. However, the mechanism involved in regulating MSC angiogenesis remains elusive. We examined the role of Nitric Oxide (NO) in this process by comparing angiogenesis by MSCs derived from wild type mice and mice lacking S-nitrosoglutathione reductase (GSNOR-/-), an enzyme that increases intracellular GSNO and enhances endogenous S-nitrosylation (a ubiquitous redox-related modification of cysteine thiol by NO) Hypothesis: Nitric oxide signaling pathways are mediated via small molecular thiols that regulate MSC angiogenesis. Method: Bone marrow-derived MSCs were isolated from wild type and GSNOR-/- mice and grown for 7 days in endothelial growth media followed by 24h in Matrigel (in vitro angiogenesis assay) in the presence of either vehicle, 15µM L-NAME (a nitric oxide synthase (NOS) inhibitor) or 500µM SNAP (a nitric oxide donor). Human (h)MSCs were similarly treated with vehicle, 15µM L-NAME and 100µM SNAP. MSC NO production and NOS expression were assessed. Results: Both types of MSCs exhibited similar levels of NO production and NOS expression, however, in the Matrigel Assay, MSCs from GSNOR-/- mice formed fewer (29.9±12.0 vs.. 50.0±15.4, p&lt;0.001) and shorter (62.8 ± 34.6 µm vs. 105.9±57.0 µm, p&lt;0.001) tubes than MSCs derived from wild type mice. L-NAME treatment normalized GSNOR-/- tube number (49.9 ± 14.8, p&lt;0.001) and length (82.8±42.5 µm, p&lt;0.001). Treatment of wild type MSCs with SNAP impaired tube formation (15.6±9.0, p&lt;0.001) and length (57.1±24.9 µm, p&lt;0.001). Similarly, hMSCs treated with SNAP exhibited impaired tube formation, both in number (25.0±4.9 vs. 49.2±8.1, p&lt;0.001) and length (81±29.1 vs. 128.0±52.5 µm, p&lt;0.001) compared to vehicle treated MSCs. L-NAME had no effect on hMSCs . Conclusion: These findings are consistent with a paradoxical negative influence of NO/GSNO on vascular formation by MSCs and reveal a novel mechanism whereby NO/GSNO inhibits MSC dependent angiogenesis. These findings have implications for disease states characterized by excessive or dysregulated angiogenesis; moreover NO deficient environments may trigger MSC mediated angiogenesis.

Nitric oxide modulates apomorphine-induced recognition memory deficits in rats

Nitric oxide (NO) is an important intracellular messenger in the brain. The implication of NO in schizophrenia is well documented although it is not yet clear whether net over or underproduction of NO is typical of this disease. In line with this, either NO donors or NO synthase (NOS) inhibitors were found to abolish psychotomimetic effects, including cognition deficits, produced by N-methyl-d-aspartate (NMDA) receptor hypofunction. In addition, there is poor experimental evidence concerning the efficacy of NO to modulate memory deficits produced by dopamine (DA) dysfunction. The present study was designed to investigate the ability of NO modulators (NO donors and NOS inhibitors to reverse recognition memory impairments produced by the DA D1/D2 mixed receptor agonist apomorphine in rats. For these studies, the novel object recognition test (NORT) was used as the memory test. Apomorphine (0.05, 0.1, 0.5 and 1.0mg/kg), dose-dependently, disrupted performance in this recognition memory procedure in rats. The NO donors molsidomine (2.0 and 4.0mg/kg) and SNP (0.3 and 1.0mg/kg), reversed the impairing effects of apomorphine (1.0mg/kg) in the NORT. Administration of the NOS inhibitors L-NAME (1.0 and 3.0mg/kg) or 7-NI (1.0 and 3.0mg/kg) produced similar results. The present findings indicate a) that apomorphine dose-dependently impaired recognition memory and b) that a cognitive deficit produced by DA dysfunction is sensitive to NO.

Up regulation of nitric oxide synthase–nitric oxide system in the testis of rats undergoing autoimmune orchitis

BackgroundMale reproductive tract infection and inflammation are important aetiological factors of infertility. Experimental Autoimmune Orchitis (EAO) is a model of chronic inflammation useful to study mechanisms of inflammatory reactions leading to testicular impairment. EAO is characterised by interstitial cell infiltrate of lymphomonocytes, producers of pro-inflammatory cytokines involved in germ cell apoptosis. Nitric oxide (NO), a free radical promoting immune cell activation and apoptosis, is synthesised by conversion of l-arginine to l-citrulline catalysed by NO synthase (NOS). The NOS isoforms are: constitutively endothelial (e) and neuronal (n) NOS and inducible (i) NOS. ObjectivesAlthough the NO–NOS system was found to be up-regulated by pro-inflammatory mediators in immune and non immune testicular cells, data on its regulation in chronic inflammatory states is lacking. Methods and resultsEAO was induced in rats by active immunisation with spermatic antigens and adjuvants; control (C) rats were injected with adjuvants. Untreated normal (N) rats were also studied. We demonstrated that iNOS, eNOS and nNOS was mainly expressed by interstitial cells in N and C rats and that in EAO NOS was up-regulated and also expressed by tubular cells. Constitutive and inducible NOS content (Western blot) as well as NO production and activity increased in the testis of rats with EAO. iNOS content and activity were selectively up-regulated in the testis of rats with orchitis. Flow cytometric analysis of NOS isoforms in testicular macrophages (M) showed that the percentage of ED1+ED2− and ED1+ED2+ M subsets, expressing constitutive and iNOS isoforms was significantly higher in EAO, but no change in the percentage of ED1−ED2+ resident M was observed compared to C rats. M from EAO rats also released more NO than C and N rats. ConclusionsIn testis of rats with EAO, NO–NOS system was up-regulated and both testicular M and cells from seminiferous tubules contributed to NO increase. NO over production in orchitis was generated mainly by increased iNOS content and activity.

Is preconditioning by oxytocin administration mediated by iNOS and/or mitochondrial KATP channel activation in the in vivo anesthetized rabbit heart?

AimsOxytocin (OXT) pretreatment protects the heart during ischemia–reperfusion injury by activating ATP-dependent potassium (KATP) channels. The aim of the current study was to elucidate the roles of nitric oxide synthaseNOS and myocardial biochemistry in the cardioprotective effects of OXT and ischemic preconditioning (IPC). Main methodsMale New Zealand White anesthetized rabbits (13 groups) were subjected to 30min of occlusion of the left coronary artery and 120min of reperfusion with or without IPC. Key findingsIPC (1cycle), OXT (0.03μg/kg, i.p.) or IPC+OXT yield significant infarct size reductions (21.8±1.5%, 20.5±1.2% and 19.4±1.4%, respectively, versus 38.9±3.5% in the S-CONT group; P<0.01) and antiarrhythmic effects, including VF (0%, 0% and 0%, versus 50% in S-CONT group; P<0.05) sustained VT (13%, 13% and 13%, versus 100% in S-CONT group; P<0.005) and other arrhythmias (25%, 13% and 25%, versus 100% in S-CONT group; P<0.005, P<0.01 and P<0.005, respectively). Atosiban (ATO, a selective OXT receptor antagonist), 5-HD and l-NAME (a nonspecific NOS inhibitor) abolished the beneficial effects of IPC and OXT, suggesting that the benefits are achieved via selective activation of OXT receptors, mitochondrial KATP channels and NO. An iNOS inhibitor (1400W) blocked the beneficial effects of IPC but not OXT. The IPC, OXT, IPC+OXT and 1400W+OXT interventions significantly preserved ATP levels in the heart. SignificanceThis study demonstrates similarities between acute OXT pretreatment and IPC in terms of infarct size reduction, antiarrhythmic activity, and metabolic status.

Enhanced Nitric Oxide Synthase‐Nitric Oxide (NOS‐NO) signaling underlies sildenafil amelioration of cyclosporine‐induced nephrotoxicity in rats

Studies have demonstrated that the potent immunosuppressive effect of cyclosporine A (CsA) is associated with severe nephrotoxicity. However, the mechanism(s) of CsA‐mediated nephrotoxicity are understudied. This study aimed to investigate the possible protective effect of sildenafil on CsA‐induced nephrotoxicity and whether this effect is mediated via enhanced NOS‐NO signaling. Wistar rats were administered sildenafil (5 mg/kg, p.o) for 21 days alone or in combination with CsA 20 mg/kg, s.c. or with the non‐selective NOS inhibitor L‐NAME; 10 mg/kg, i.p. or both. Control groups were treated with either vehicle, CsA or L‐NAME. Renal functions (serum urea and creatinine, creatinine clearance and albumin‐creatinine ratio), indices of antioxidant activities (catalase, reduced glutathione and malondialdehyde), total nitrate/nitrite (NOx) were measured. Apoptotic and inflammatory markers (caspase‐3 and TNF‐alpha) were measured by. CsA administration for 21 days resulted in significant renal oxidative stress, deterioration of renal functions, reduction of NOx levels, marked apoptotic effect and elevation of nephritic TNF‐alpha expression. Concomitant treatment with sildenafil significantly improved CsA‐renal dysfunction, elevated NOx levels and reduced nephritic oxidative stress. Furthermore, sildenafil attenuated CsA‐evoked apoptosis and TNF‐alpha expression. Interestingly, L‐NAME reversed sildenafil protective effects against CsA‐mediated nephrotoxicity. Taken together, the current study highlights the clinically significant sildenafil protective effects against CsA‐induced nephrotoxicity that might be mediated via enhanced NOS‐NO signaling.

Inhibition of germinal vesicle breakdown by antioxidants and the roles of signaling pathways related to nitric oxide and cGMP during meiotic resumption in oocytes of a marine worm

In mammalian oocytes, cAMP elevations prevent the resumption of meiotic maturation and thereby block nuclear disassembly (germinal vesicle breakdown (GVBD)), whereas nitric oxide (NO) and its downstream mediator cGMP can either inhibit or induce GVBD. Alternatively, some invertebrate oocytes use cAMP to stimulate, rather than inhibit, GVBD, and in such cases, the effects of NO/cGMP signaling on GVBD remain unknown. Moreover, potential interactions between NO/cGMP and AMP-activated kinase (AMPK) have not been assessed during GVBD. Thus, this study analyzed intraoocytic signaling pathways related to NO/cGMP in a marine nemertean worm that uses cAMP to induce GVBD. For such tests, follicle-free nemertean oocytes were stimulated to mature by seawater (SW) and cAMP elevators. Based on immunoblots and NO assays of maturing oocytes, SW triggered AMPK deactivation, NO synthase (NOS) phosphorylation, and an NO elevation. Accordingly, SW-induced GVBD was blocked by treatments involving the AMPK agonist AICAR, antioxidants, the NO scavenger carboxy-PTIO, NOS inhibitors, and cGMP antagonists that target the NO-stimulated enzyme, soluble guanylate cyclase (sGC). Conversely, SW solutions combining NO/cGMP antagonists with a cAMP elevator restored GVBD. Similarly, AICAR plus a cAMP-elevating drug reestablished GVBD while deactivating AMPK and phosphorylating NOS. Furthermore, sGC stimulators and 8-Br-cGMP triggered GVBD. Such novel results indicate that NO/cGMP signaling can upregulate SW-induced GVBD and that cAMP-elevating drugs restore GVBD by overriding the inhibition of various NO/cGMP downregulators, including AMPK. Moreover, considering the opposite effects of intraoocytic cAMP in nemerteans vs mammals, these data coincide with previous reports that NO/cGMP signaling blocks GVBD in rats.

Nitric oxide (NO) — Production and regulation of insulin secretion in islets of freely fed and fasted mice

Production of nitric oxide through the action of nitric oxide synthase (NOS) has been detected in the islets of Langerhans. The inducible isoform of NOS (iNOS) is induced by cytokines and might contribute to the development of type-1 diabetes, while the constitutive isoform (cNOS) is thought to be implicated in the physiological regulation of insulin secretion. In the present study we have detected and quantified islet cNOS- and iNOS-derived NO production concomitant with measuring its influence on insulin secretion in the presence of different secretagogues: glucose, l-arginine, l-leucine and α-ketoisocaproic acid (KIC) both during fasting and freely fed conditions. In intact islets from freely fed mice both cNOS- and iNOS-activity was greatly increased by glucose (20mmol/l). Fasting induced islet iNOS activity at both physiological (7mmol/l) and high (20mmol/l) glucose concentrations. NOS blockade increased insulin secretion both during freely fed conditions and after fasting. l-arginine stimulated islet cNOS activity and did not affect islet iNOS activity. l-leucine or KIC, known to enter the TCA cycle without affecting glycolysis, did not affect either islet cNOS- or iNOS activity. Accordingly, insulin secretion stimulated by l-leucine or KIC was unaffected by addition of l-NAME both during feeding and fasting. We conclude that both high glucose concentrations and fasting increase islet total NO production (mostly iNOS derived) which inhibit insulin secretion. The insulin secretagogues l-leucine and KIC, which do not affect glycolysis, do not interfere with the islet NO–NOS system.

Rosiglitazone reverses increased duodenal inhibitory response in spontaneously hypertensive rats

Thiazolidinediones (TZDs) including rosiglitazone (ROSI) are insulin sensitizing agents with beneficial gastrointestinal effects. However, no studies are available on TZDs effect in gastrointestinal motility. We evaluated the effects of ROSI on gastrointestinal inhibitory neurotransmission focusing on the modulatory roles of nitric oxide synthase/nitric oxide (NOS/NO) and heme oxygenase/carbon monoxide (HO/CO) pathways. Spontaneously hypertensive rats (SHR) were used as model of insulin resistance. Duodenal strips were obtained from vehicle-treated SHR, ROSI-treated SHR (5 mg kg(-1) by gavage daily per 6 weeks), and Wistar Kyoto (WKY). Inhibitory responses to electrical field stimulation (EFS) were evaluated in the presence of HO inhibitor zinc protoporphyrin IX (ZnPPIX, 10 μmol L(-1)) or NOS inhibitor N(G)-nitro-L-arginine (L-NNA, 100 μmol L(-1)), alone and in combination. Protein levels of HO and NOS isoforms were evaluated by immunohistochemistry and western blot analysis. Basal responses to EFS were significantly increased in duodenum strips from vehicle-treated SHR vs WKY. This effect was reversed in ROSI-treated SHR. The EFS-mediated relaxation was comparably reduced by ZnPPIX in WKY and SHR, but not in ROSI-treated SHR animals. The L-NNA reduced EFS response to a similar extent in WKY and ROSI -treated SHR, but its effect was significantly higher in vehicle-treated SHR. Expression of HO-1 protein was significantly lower, whereas HO-2 protein levels were unchanged in ROSI-treated SHR with respect to vehicle-treated SHR. Finally, increased levels of nNOS in vehicle-treated SHR were reduced in ROSI-treated SHR. Chronic ROSI treatment reverses increased SHR duodenal inhibitory response acting on CO and NO components.

Expression of nitric oxide and nitric oxide synthase in patients with acute massive cerebral infarction

Objective To study the expression of nitric oxide (NO) and nitric oxide synthase (NOS) in patients with acute massive cerebral infarction. Methods Sixteen patients (cerebral infarction group) with acute massive cerebral infarction were subjected to craniotomy decompression craniectomy under the anesthesia, and part of the infarct frontal lobe was removed. The normal frontal lobe from 6 cases of severe brain injury receiving the intracranial decompression served as control group. The ultrastructures of frontal lobe cells were observed under the electron microscopy. Serum NO content and NOS activity were measured. The protein expression patterns of eNOS, nNOS and iNOS were analyzed by Western blotting.Results The consciousness in 16 patients with acute massive cerebral infarction was improved after operation to varying degrees, and there were no deaths. The NO contents and NOS activities in cerebral infarction group and control groups were 85.4 and 36. 2 U/mi, and 63.2 and 37.2 μmol/L ( all P ＜ 0. 05). As compared with control group, the expression of eNOS, nNOS and iNOS in cerebral infarction group was significantly increased, especially the expression of iNOS ( all P ＜ 0. 05). Conclusion Craniotomy decompression craniectomy was the effective treatment to cure the acute massive cerebral infarction. It could reduce mortality. NO and NOS participated in and influenced the complex pathophysiological process of massive cerebral infarction. After craniotomy decompression craniectomy the regional cerebral blood was effectively improved, and NOS activity, especially iNOS, and the synthesis of NO were reduced, resulting in the reduction to neurotoxicity and improvement in clinical symptoms. Key words: Cerebral infarction; Nitric oxide synthase; Decompressive craniectomy

Amelioration effects of berberine on diabetic microendothelial injury model by the combination of high glucose and advanced glycation end products in vitro

Microvascular complications are much earlier and common in diabetes. Advanced glycation end products (AGEs), together with high glucose, play a key role in the endothelial dysfunction of diabetic vascular complications. So it is of more significance to expedite the therapies to block the formation and/or the effects of AGEs. Berberine has been showed to have anti-diabetic effects, however the effects on diabetic complications were less explored, especially the effects on the microvascular complications and the formation and pathways of AGEs which have not been reported. Therefore, the present study established an in vitro model of diabetic microendothelial (microEC) injury by the combination of high glucose and AGEs to mimic the clinical situations and examine the effects and mechanisms of berberine on high glucose-AGEs-induced microEC injuries and on the formation of AGEs. We prepared AGEs, established the high glucose-AGEs injured microEC models by MTT assay, which was further supported by significantly decreased nitric oxide (NO) release, NO synthase (NOS) and thrombomodulin production with ELISA, western blot and RT-PCR analysis. Berberine treatments showed significant improvements as indicated by significantly increased NO release, NOS and thrombomodulin production. Moreover, we also observed significant inhibition effects of berberine on AGEs formation. We concluded that the in vitro model of diabetic microEC injury could be established by the combination treatments of high glucose and AGEs, while berberine could improve the diabetic microvascular injury in vitro and inhibit the formation of AGEs, suggesting the potential clinical therapies with berberine for diabetes and its vascular complications.

Nitric oxide synthase expression in foetal placentas of cows with retained fetal membranes

The objectives of this study were to investigate relationship of retained fetal membranes (RFM) to expression of NOS and NOS mRNA and to analyze pathohistological changes and the distribution of nitric oxide synthase (NOS) in foetal placentas of cows with RFM. Twenty cows were assigned to two groups, a control group (no retained fetal membranes, NRFM, n = 10) and a diseased group (RFM, n = 10). The endpoint method was used to detect the nitric oxide (NO) content and nitric oxide synthase (NOS) activity in foetal placental tissue fluid and the fluorescent quantitation PCR was used to measure the expression of NOS mRNA. Immunohistochemistry and hematoxylin–eosin staining were used to observe pathohistological changes. Tissue from RFM cows showed fibronecrosis of the chorionic villi, and a decreased number of trophoblastic cells. The majority of trophoblastic cells displayed vacuolar degeneration. Interstitium vessels were distended and congested. Expression of induced nitric oxide synthase (iNOS) protein and iNOS mRNA was significantly higher ( P < 0.05) in the cytoplasm of placental villus trophoblastic cells in the RFM group. But expression of endothelial nitric oxide synthase (eNOS) protein and eNOS mRNA was significantly lower ( P < 0.05) in the RFM group. The NO content and NOS activity of cows with RFM were significantly higher ( P < 0.05). A high expression of iNOS protein and iNOS mRNA in the cow foetal placenta could produce high content of NO, which might inhibit uterine contraction. So over expression of iNOS protein and iNOS mRNA might be an important agent of retained fetal membranes in cows, and it may be a potential diagnosis biomarker.

Two enzymatic sources of nitric oxide in different organs of apple plant

Nitric oxide production, nitric oxide synthase (NOS) and mitochondrial nitrite-reducing activities in roots, leaves and stems of different developmental stages were investigated, using potted 3-year-old apple (Malus domestica Borkh.) trees. The arginine-dependent NOS activity is sensitive to NOS inhibitor L-NAME and aminoguanidine (AG), with L-NAME being more effective than AG. Endogenous NO production, NOS and mitochondrial nitrite-reducing activities are predominately presented in young leaves and especially in young white roots and young stems. Root and stem mitochondria can reduce nitrite to nitric oxide at the expense of NADH, however, this mitochondrial nitrite-reducing activity is absent in leaves.

Interaction of endothelin-1 and nitric oxide pathways in human tubular epithelial cells under the influence of cyclosporine-A

Background: The exact mechanism of cyclosporine (CsA) nephrotoxicity has not been clarified. In this study, we investigated the effect of pharmacological doses of CsA on the production of nitric oxide synthases (NOSs) and endothelin (ET) receptors (ETR-A, ETR-B), in human tubular cells [human kidney (HK)-2], to identify any implication of these pathways in CsA nephrotoxicity. Methods: Human tubular epithelial cells (HK-2) were cultured in the presence of CsA at various concentrations (0–1000 ng/mL). Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine mRNA synthesis of NOSs (eNOS, iNOS) and ET receptors (ETR-A, ETR-B) and western blot analysis for the subsequent proteins. Results: A dose-dependent induction of synthesis of NO synthases eNOS and iNOS and ET receptors ETR-A and ETR-B was observed, even at therapeutic doses of CsA. An interaction between NO and ET-1 systems under the influence of CsA was also observed. Blockage of NO production was followed by down-regulation of ETR-B whereas blockade of ET pathway with ET receptor antagonists was followed by down-regulation of eNOS expression. Conclusion: CsA induces NOSs as well as ET receptor mRNA and protein synthesis in tubular epithelial cells. The up-regulation of NO and ET-1 pathways is probably implicated in the nephrotoxic action of CsA, whereas an interplay between ETR-B and eNOS seems to be involved.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamus in vitro

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

Carbon monoxide and nitric oxide modulate hyperosmolality-induced oxytocin secretion by the hypothalamusin vitro

OT (oxytocin) is secreted from the posterior pituitary gland, and its secretion has been shown to be modulated by NO (nitric oxide). In rats, OT secretion is also stimulated by hyperosmolarity of the extracellular fluid. Furthermore, NOS (nitric oxide synthase) is located in hypothalamic areas involved in fluid balance control. In the present study, we evaluated the role of the NOS/NO and HO (haem oxygenase)/CO (carbon monoxide) systems in the osmotic regulation of OT release from rat hypothalamus in vitro. We conducted experiments on hypothalamic fragments to determine the following: (i) whether NO donors and NOS inhibitors modulate OT release and (ii) whether the changes in OT response occur concurrently with changes in NOS or HO activity in the hypothalamus. Hyperosmotic stimulation induced a significant increase in OT release that was associated with a reduction in nitrite production. Osmotic stimulation of OT release was inhibited by NO donors. NOS inhibitors did not affect either basal or osmotically stimulated OT release. Blockade of HO inhibited both basal and osmotically stimulated OT release, and induced a marked increase in NOS activity. These results indicate the involvement of CO in the regulation of NOS activity. The present data demonstrate that hypothalamic OT release induced by osmotic stimuli is modulated, at least in part, by interactions between NO and CO.

Reassessing the risk of hemodilutional anemia: Some new pieces to an old puzzle

Clinical studies demonstrate that anemia increases the risk of morbidity and mortality. Tissue hypoxia is an attractive but incompletely characterized candidate mechanism of anemia-induced organ injury. Physiological responses that optimize tissue oxygen delivery (nitric oxide synthase-NOS) and promote cellular adaptation to tissue hypoxia (hypoxia inducible factor-HIF) may reduce the risk of hypoxic organ injury and thereby improve survival during anemia. The presence of vascular diseases would likely impair the efficacy of these physiological mechanisms, increasing the risk of anemia-induced organ injury. In all cases, biological signals that indicate the activation of these adaptive mechanisms could provide an early and treatable warning signal of impending anemia-induced organ injury. Thus, we review the evidence for tissue hypoxia during acute hemodilutional anemia and also explore the novel hypothesis that methemoglobin, a measurable byproduct of increased NOS-derived nitric oxide (NO), may serve as a biomarker for "anemic stress". Published peer-reviewed studies provided the main source of information. Data from experimental studies were reassessed to derive the relationship between hemodilution (reduced hemoglobin concentration) and increased methemoglobin levels. Active physiological mechanisms (sympathetic nervous system) are required to maintain optimal tissue oxygen delivery during hemodilutional anemia. Despite these responses, tissue hypoxia occurs during acute hemodilution, as demonstrated by a decrease in tissue PO(2) and an increase in hypoxic cellular responses (NOS, HIF). Optimal tissue oxygen delivery may be compromised further when cardiovascular responses are impaired. The positive correlation between decreased hemoglobin concentration (Hb) and an increase in methemoglobin levels in acutely anemic animals supports the hypothesis that anemia-induced increases in tissue NOS activity could promote methemoglobin formation. Methemoglobin may be a measurable byproduct of NO-mediated Hb oxidation. Evidence continues to demonstrate that anemia increases morbidity and mortality, possibly via hypoxic mechanisms. A potential strategy for assessing "anemic stress" was derived from experimental data based on a readily measurable biomarker, methemoglobin. New methods for measuring real-time hemoglobin and methemoglobin levels in patients may provide the basis to translate this idea into clinical practice. Further mechanistic studies are required to determine if the impact of reduced tissue oxygen delivery and activation of hypoxic cellular mechanism can be linked to measurable changes in biomarkers and clinical outcomes in acutely anemic patients.