Nitric Oxide Synthase In Response Research Articles

A simple protocol for isolating microglia from adult mouse brain.

Although microglia are activated in adult and aged brains resulting in neurodegenerative and neuroinflammatory disorders, most of the cell culture studies on microglia deal with neonatal microglia because of ease of isolation. Microglia could be isolated from adult brains, but it requires separation by density gradient centrifugation, magnetic beads, etc. Here, we describe a simple protocol of isolating highly purified microglia from adult mouse brains. Our protocol involves dilution with sterile PBS or media, regular centrifugation, and plating on poly-D-lysine-coated flasks. These adult microglia expressed the inducible nitric oxide synthase in response to preformed α-syn fibril, an etiological reagent of Parkinson's disease, and bacterial lipopolysaccharides, one of the prototype proinflammatory stimuli. Moreover, these adult microglia exhibited phagocytosis, which was stimulated by LPS treatment. These results suggest that adult microglia isolated by our procedure are functional and that these adult microglia could be used for studies related to neurodegenerative disorders.

Amomum muricarpum Elmer 추출물의 Lipopolysachharide에 의한 염증반응 조절 기전 분석

Background: Identifying new natural products to controlling inflammatory responses involved in screening of Asian native plants. The screening revealed a potential candidate, Amomum muricarpum Elmer, which is a traditional medicinal plant for gastrointestinal diseases in Vietnam; however, its scientific basis remains unknown, its scientific basis remain unknown.BRMethods and Results: A. muricarpum extract suppressed lipopolysaccharide (LPS)-induced the protein expression of inflammatory mediators such as interleukine (IL)-1β and cyclooxygenase-2 in mouse-derived RAW264.7 macrophages. The mRNA expression levels of IL-6, IL-1β, tumor necrosis factor-α, and inducible nitric oxide synthase in response to LPS were reduced by A. muricarpum extract. Additionally, A. muricarpum extract inhibited phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa B (NF-κB). This result indicates A. muricarpum suppressed the expression of inflammatory cytokines and mediators by inhibiting signal transducer and STAT3 and NF-κB activation.BRConclusions: This is the first scientific evidence to report that A. muricarpum suppresses inflammatory signaling pathways and can potentially manage excessive and chronic inflammatory responses.

Regulation of Lung Macrophage Activation and Oxidative Stress Following Ozone Exposure by Farnesoid X Receptor.

Inflammatory macrophages are known to contribute to ozone toxicity. Farnesoid X receptor (FXR) is a nuclear receptor involved in regulating bile acid and lipid homeostasis; it also exerts anti-inflammatory activity by suppressing macrophage NF-κB. Herein, we analyzed the role of FXR in regulating macrophage activation in the lung following ozone exposure. Treatment of wild-type (WT) mice with ozone (0.8 ppm, 3 h) resulted in increases in proinflammatory (F4/80+CD11c+CD11b+Ly6CHi) and anti-inflammatory (F4/80+CD11c+CD11b+Ly6CLo) macrophages in the lung. The accumulation of proinflammatory macrophages was increased in FXR-/- mice compared with WT mice; however, anti-inflammatory macrophage activation was blunted as reflected by reduced arginase and mannose receptor expression, a response correlated with decreased Nur77. This was associated with prolonged oxidative stress, as measured by 4-hydroxynonenal-modified proteins in the lung. Loss of FXR was accompanied by protracted increases in lung NF-κB activity and its target, inducible nitric oxide synthase in response to ozone. Levels of Tnf-α, Il-1β, Ccr2, Ccl2, Cx3cr1, and Cx3cl1 were also increased in lungs of FXR-/- relative to WT mice; conversely, genes regulating lipid homeostasis including Lxrα, Apoe, Vldlr, Abcg1, and Abca1 were downregulated, irrespective of ozone exposure. In FXR-/- mice, ozone caused an increase in total lung phospholipids, with no effect on SP-B or SP-D. Dyslipidemia was correlated with blunting of ozone-induced increases in positive end-expiratory pressure-dependent quasi-static pressure volume curves indicating a stiffer lung in FXR-/- mice. These findings identify FXR as a regulator of macrophage activation following ozone exposure suggesting that FXR ligands may be useful in mitigating inflammation and oxidative stress induced by pulmonary irritants.

Chronic Isolation Stress Affects Subsequent Crowding Stress-Induced Brain Nitric Oxide Synthase (NOS) Isoforms and Hypothalamic-Pituitary-Adrenal (HPA) Axis Responses

The nitric oxide (NO) pathway in the brain is involved in response to psychosocial stressors. The aim of this study was to elucidate the role of nNOS and iNOS in the prefrontal cortex (PFC), hippocampus (HIP), and hypothalamus (HYPO) during social isolation stress (IS), social crowding stress (CS), and a combined IS + CS. In the PFC, 3 days of CS increased iNOS but not nNOS protein level. In the HIP and HYPO, the levels of nNOS and iNOS significantly increased after 3 days of CS. In the PFC, IS alone (11 days) enhanced iNOS protein level following 3 days of CS and increased nNOS level in the HIP and HYPO after 14 days of CS. By contrast, in the HIP, IS abolished the subsequent CS-induced increase in nNOS in the HIP and strongly elevated iNOS level after 7 days of CS. In the HYPO, prior IS inhibited nNOS protein level induced by subsequent CS for 3 days, but increased nNOS protein level after longer exposure times to CS. Isolation stress strongly upregulated plasma interleukin-1β (IL-1β) and adrenocorticotropic hormone (ACTH) levels while corticosterone (CORT) level declined. We show that the modulatory action of the NO pathway and ACTH/CORT adaptation to chronic social isolation stress is dependent on the brain structure and nature and duration of the stressor. Our results indicate that isolation is a robust natural stressor in social animals; it enhances the NO pathway in the PFC and abolishes subsequent social CS-induced NOS responses in the HIP and HYPO.

Emerging new role of NFAT5 in inducible nitric oxide synthase in response to hypoxia in mouse embryonic fibroblast cells

We previously described the protective role of the nuclear factor of activated T cells 5 (NFAT5) during hypoxia. Alternatively, inducible nitric oxide synthase (iNOS) is also induced by hypoxia. Some evidence indicates that NFAT5 is essential for the expression of iNOS in Toll-like receptor-stimulated macrophages and that iNOS inhibition increases NFAT5 expression in renal ischemia-reperfusion. Here we studied potential NFAT5 target genes stimulated by hypoxia in mouse embryonic fibroblast (MEF) cells. We used three types of MEF cells associated with NFAT5 gene: NFAT5 wild type (MEF-NFAT5+/+), NFAT5 knockout (MEF-NFAT5-/-), and NFAT5 dominant-negative (MEF-NFAT5Δ/Δ) cells. MEF cells were exposed to 21% or 1% O2 in a time course curve of 48 h. We found that, in MEF-NFAT5+/+ cells exposed to 1% O2, NFAT5 was upregulated and translocated into the nuclei, and its transactivation domain activity was induced, concomitant with iNOS, aquaporin 1 (AQP-1), and urea transporter 1 (UTA-1) upregulation. Interestingly, in MEF-NFAT5-/- or MEF-NFAT5Δ/Δ cells, the basal levels of iNOS and AQP-1 expression were strongly downregulated, but not for UTA-1. The upregulation of AQP-1, UTA-1, and iNOS by hypoxia was blocked in both NFAT5-mutated cells. The iNOS induction by hypoxia was recovered in MEF-NFAT5-/- MEF cells, when recombinant NFAT5 protein expression was reconstituted, but not in MEF-NFAT5Δ/Δ cells, confirming the dominant-negative effect of MEF-NFAT5Δ/Δ cells. We did not see the rescue effect on AQP-1 expression. This work provides novel and relevant information about the signaling pathway of NFAT5 during responses to oxygen depletion in mammalian cells and suggests that the expression of iNOS induced by hypoxia is dependent on NFAT5.

Expression of human inducible nitric oxide synthase in response to cytokines is regulated by hypoxia-inducible factor-1

The production of nitric oxide (NO) by inducible NO synthase (iNOS) and the regulation of gene expression by hypoxia-inducible factors (HIFs) are important for many aspects of human cell biology. However, little is known about whether iNOS expression is controlled by HIFs in human cells. Stimulation of A549 human lung epithelial cells with cytokines (TNF, IL-1 and IFNγ) increased the nuclear accumulation of HIF-1 in normoxic conditions. Activation of HIF-1 by hypoxia or CoCl2 was not sufficient to induce iNOS expression. However, pharmacological inhibition of HIF-1 reduced the induction of iNOS expression in A549 cells and primary human astrocytes. Moreover, elimination of HIF-1α expression and activity by CRISPR/Cas9 gene editing significantly reduced the induction of human iNOS gene promoter, mRNA and protein expression by cytokine stimulation. Three putative hypoxia response elements (HRE) are present within the human iNOS gene promoter and elimination of an HRE at −4981 bp reduced the induction of human iNOS promoter activity in response to cytokine stimulation. These findings establish an important role for HIF-1α in the induction of human iNOS gene expression in response to cytokine stimulation.

The Effects of Magnesium Sulfate on the Inflammatory Response of Placentas Perfused With Lipopolysaccharide: Using the Ex Vivo Dual-Perfused Human Single-Cotyledon Model.

Multiple mechanisms have been proposed for the neuroprotective effects of magnesium sulfate (MgSO4). We aimed to examine the effects of lipopolysaccharide (LPS) and MgSO4 on the placental expression of nuclear factor κ light chain enhancer of activated B cells (NF-κB), interleukin (IL) 6, adrenocorticotropic hormone (ACTH), and nitric oxide synthase (NOS); all known to participate in the inflammatory cascade. Placentas were obtained and selected cotyledons cannulated and dually perfused ex vivo. Placentas were perfused with 4 perfusion protocols: culture medium (M-199; controls), LPS (1 μg/mL), MgSO4 (6 g/dL), and LPS + MgSO4. Each perfusion experiment continued for 3 hours. Sixteen perfusion experiments were analyzed, 4 separate placentas were studied for each protocol. The protein levels in the perfused cotyledons were studied by Western blot analysis and compared between the groups. Interleukin 6 levels were studied in the maternal and fetal perfusate. The expression of NF-κB p65, IL-6, ACTH, and NOS proteins levels were significantly increased in placentas perfused with LPS as compared to placentas perfused with M-199, MgSO4 ( P < .01 for all). Placentas perfused with LPS+ MgSO4 had similar proteins levels as in the controls and MgSO4 groups. Lipopolysaccharide significantly increased IL-6 levels in maternal perfusate. In the human placenta, MgSO4 blocks the increase in the proteins levels of NF-κB, IL-6, ACTH, and NOS in response to inflammatory stimuli. Magnesium sulfate attenuates excessive placental inflammatory response. The decrease in placental ACTH levels following perfusion with MgSO4 may point to an additional non-anti-inflammatory mechanism of MgSO4.

A Bioprocessed Polysaccharide from Lentinus edodes Mycelia Cultures with Turmeric Protects Chicks from a Lethal Challenge of Salmonella Gallinarum

Our previous studies demonstrated that a bioprocessed polysaccharide (BPP) isolated from Lentinus edodes mushroom mycelia cultures supplemented with black rice bran can protect mice against Salmonella lipopolysaccharide-induced endotoxemia and reduce the mortality from Salmonella Typhimurium infection through upregulated T-helper 1 immunity. Here, we report that a BPP from L. edodes mushroom mycelia liquid cultures supplemented with turmeric (referred to as BPP-turmeric) alters chicken macrophage responses against avian-adapted Salmonella Gallinarum and protects chicks against a lethal challenge from Salmonella Gallinarum. In vitro analyses revealed that the water extract of BPP-turmeric (i) changed the protein expression or secretion profile of Salmonella Gallinarum, although it was not bactericidal, (ii) reduced the phagocytic activity of the chicken-derived macrophage cell line HD-11 when infected with Salmonella Gallinarum, and (iii) significantly activated the transcription expression of interleukin (IL)-1β, IL-10, tumor necrosis factor α, and inducible nitric oxide synthase in response to various Salmonella infections, whereas it repressed that of IL-4, IL-6, interferon-β, and interferon-γ. We also found that BPP-turmeric (0.1 g/kg of feed) as a feed additive provided significant protection to 1-day-old chicks infected with a lethal dose of Salmonella Gallinarum. Collectively, these results imply that BPP-turmeric contains biologically active component(s) that protect chicks against Salmonella Gallinarum infection, possibly by regulating macrophage immune responses. Further studies are needed to evaluate the potential efficacy of BPP-turmeric as a livestock feed additive for the preharvest control of fowl typhoid or foodborne salmonellosis.

Antioxidant diet and sex interact to regulate NOS isoform expression and glomerular mesangium proliferation in Zucker diabetic rat kidney

Oxidative stress contributes substantially to the pathophysiology of diabetic nephropathy (DN). Consumption of an antioxidant-fortified (AO) diet from an early age prevents or delays later development of DN in the Zucker rat female with type 2 diabetes. We hypothesize this is due to effects on mesangial matrix and renal nitric oxide synthase (NOS) distribution and to sex-specific differences in NOS responses in the diabetic kidney. Total glomerular tuft area (GTA) and PAS-positive tuft area (PTA), endothelial (e), neuronal (n) and inducible (i) NOS were quantified in males and females on AO or regular (REG) diet at 6 and 20 weeks of age. eNOS was observed in glomeruli and tubules. nNOS predominantly localized to tubular epithelium in both cortex and medulla. iNOS was expressed in proximal and distal tubules and collecting ducts. Sex, diabetes duration and AO diet affected the distribution of the three isoforms. GTA and PTA increased with duration of hyperglycemia and showed a negative correlation with renal levels of all NOS isoforms. AO diet in both genders was associated with less PAS-positive staining and less mesangial expansion than the REG diet, an early increase in cortical iNOS in males, and sex-specific changes in cortical eNOS at 20 weeks. These effects of AO diet may contribute to sex-specific preservation of renal function in females.

The F-BAR Protein NOSTRIN Dictates the Localization of the Muscarinic M3 Receptor and Regulates Cardiovascular Function.

Endothelial dysfunction is an early event in cardiovascular disease and characterized by reduced production of nitric oxide (NO). The F-BAR protein NO synthase traffic inducer (NOSTRIN) is an interaction partner of endothelial NO synthase and modulates its subcellular localization, but the role of NOSTRIN in pathophysiology in vivo is unclear. We analyzed the consequences of deleting the NOSTRIN gene in endothelial cells on NO production and cardiovascular function in vivo using NOSTRIN knockout mice. The levels of NO and cGMP were significantly reduced in mice with endothelial cell-specific deletion of the NOSTRIN gene resulting in diastolic heart dysfunction. In addition, systemic blood pressure was increased, and myograph measurements indicated an impaired acetylcholine-induced relaxation of isolated aortic rings and resistance arteries. We found that the muscarinic acetylcholine receptor subtype M3 (M3R) interacted directly with NOSTRIN, and the latter was necessary for correct localization of the M3R at the plasma membrane in murine aorta. In the absence of NOSTRIN, the acetylcholine-induced increase in intracellular Ca(2+) in primary endothelial cells was abolished. Moreover, the activating phosphorylation and Golgi translocation of endothelial NO synthase in response to the M3R agonist carbachol were diminished. NOSTRIN is crucial for the localization and function of the M3R and NO production. The loss of NOSTRIN in mice leads to endothelial dysfunction, increased blood pressure, and diastolic heart failure.

Cyclooxygenase inhibition improves endothelial vasomotor dysfunction of visceral adipose arterioles in human obesity

ObjectiveThe purpose of this study was to determine whether cyclooxygenase inhibition improves vascular dysfunction of adipose microvessels from obese humans.Design and MethodsIn 20 obese subjects (age 37±12 yrs, BMI 47±8 kg/m2) we collected subcutaneous and visceral fat during bariatric surgery and characterized adipose depot-specific gene expression, endothelial cell phenotype, and microvascular function. Vasomotor function was assessed in response to endothelium-dependent agonists using videomicroscopy of small arterioles from fat.ResultsArterioles from visceral fat exhibited impaired endothelium-dependent, acetylcholine-mediated vasodilation, compared to the subcutaneous depot (p<0.001). Expression of mRNA transcripts relevant to the cyclooxygenase pathway were upregulated in visceral compared to subcutaneous fat. Pharmacological inhibition of cyclooxygenase with indomethacin improved endothelium-dependent vasodilator function of arterioles from visceral fat by 2-fold (p=0.01), whereas indomethacin had no effect in the subcutaneous depot. Indomethacin increased activation via serine-1177 phosphorylation of endothelial nitric oxide synthase in response to acetylcholine in endothelial cells from visceral fat. Inhibition of endothelial nitric oxide synthase with Nω-nitro-L-arginine methyl ester abrogated the effects of cyclooxygenase-inhibition suggesting that vascular actions of indomethacin were related to increased nitric oxide bioavailability.ConclusionsOur findings suggest that cyclooxygenase-mediated vasoconstrictor prostanoids partly contribute to endothelial dysfunction of visceral adipose arterioles in human obesity.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Kinetic Study on the Effects of Extremely Low Frequency Electromagnetic Field on Catalase, Cytochrome P450 and Inducible Nitric Oxide Synthase in Human HaCaT and THP-1 Cell Lines

Extremely low frequency electromagnetic fields (ELF-EMF) have been found to produce a variety of biological effects. These effects of ELF-EMF depend upon frequency, amplitude, and length of exposure, and are also related to intrinsic susceptibility and responsiveness of different cell types. Although the mechanism of this interaction is still obscure, ELF-EMF can influence cell proliferation, differentiation, cell cycle, apoptosis, DNA replication and protein expression. The aim of this study was to estimate various kinetic constants of catalase, cytochrome P450 and inducible nitric oxide synthase in response to ELF-EMF exposure in human HaCaT and THP-1 cell lines. In order to evaluate the effect of ELF-EMF on the modulation of cellular responses to an inflammatory stimulus, both cell lines were treated with lipopolysaccharide. To the best of our knowledge there is no available report on such type of kinetic study of selected enzymes in response to ELF-EMF in these cell lines. Therefore, the current study may reveal novel mechanism of ELFEMF biological interaction with the enzymological and hormonal systems of living organisms. These new insights may be important for ELF-EMF application particularly for wound healing, tissue regeneration, Parkinson's and Alzheimer's diseases.

Caveolin 1 is required for the activation of endothelial nitric oxide synthase in response to 17beta-estradiol.

Evidence suggests that estrogen mediates rapid endothelial nitric oxide synthase (eNOS) activation via estrogen receptor-a (ERalpha) within the plasma membrane of endothelial cells (EC). ERalpha is known to colocalize with caveolin 1, the major structural protein of caveolae, and caveolin 1 stimulates the translocation of ERalpha to the plasma membrane. However, the role played by caveolin 1 in regulating 17beta-estradiol-mediated NO signaling in EC has not been adequately resolved. Thus, the purpose of this study was to explore how 17beta-estradiol stimulates eNOS activity and the role of caveolin 1 in this process. Our data demonstrate that modulation of caveolin 1 expression using small interfering RNA or adenoviral gene delivery alters ERalpha localization to the plasma membrane in EC. Further, before estrogen stimulation ERalpha associates with caveolin 1, whereas stimulation promotes a pp60(Src)-mediated phosphorylation of caveolin 1 at tyrosine 14, increasing ERalpha-PI3 kinase interactions and disrupting caveolin 1-ERalpha interactions. Adenoviral mediated overexpression of a phosphorylation-deficient mutant of caveolin (Y14FCav) attenuated the ERalpha/PI3 kinase interaction and prevented Akt-mediated eNOS activation. Furthermore, Y14FCav overexpression reduced eNOS phosphorylation at serine1177 and decreased NO generation after estrogen exposure. Using a library of overlapping peptides we identified residues 62-73 of caveolin 1 as the ERalpha-binding site. Delivery of a synthetic peptide based on this sequence decreased ERalpha plasma membrane translocation and reduced estrogen-mediated activation of eNOS. In conclusion, caveolin 1 stimulates 17beta-estradiol-induced NO production by promoting ERalpha to the plasma membrane, which facilitates the activation of the PI3 kinase pathway, leading to eNOS activation and NO generation.

Histone Eviction During Hypoxia

Histones are removed and then replaced at the promoter of the gene encoding endothelial nitric oxide synthase in response to hypoxia.

Dietary fat differentially influences regulatory endothelial function during the postprandial state in patients with metabolic syndrome: From the LIPGENE study

Objective To investigate whether endothelium-dependent vasomotor function and plasma levels of cellular adhesion molecules are affected by diets with different fat quantity and quality during the postprandial state in subjects with the metabolic syndrome (MetS). Methods Patients were randomly assigned to one of four isoenergetic diets distinct in fat quantity and quality: high-SFA (HSFA); high-MUFA (HMUFA) and two low-fat, high-complex carbohydrate (LFHCC) diets, supplemented with 1.24 g/day of long chain n-3 PUFA (LC n-3 PUFA) or placebo for 12 weeks each. Flow-associated vasodilatation of the brachial artery and postprandial plasma levels of total nitrites, nitric oxide (NO) synthase, soluble intercellular cell adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and P-selectin were assessed post-intervention. Results Post-intervention postprandial flow-associated vasodilatation was significantly higher after the HMUFA diet ( P < 0.05) compared to subjects adhering to the other three diets. Consistently, the postprandial NO synthase response significantly increased during the HMUFA compared with the HSFA and LFHCC (placebo) diets. Postprandial sICAM-1 levels were lower during the HMUFA than during the HSFA and LFHCC n-3 diets. Conclusions Our data support the notion that the HMUFA diet improves postprandial endothelial cell function and decreases postprandial plasma sICAM-1 concentrations in patients with the MetS. These findings suggest that the postprandial state is important for understanding possible cardio-protective effects associated with the Mediterranean diet particularly in subject with the MetS.

Identification of leishmania fructose-1,6-bisphosphate aldolase as a novel activator of host macrophage Src homology 2 domain containing protein tyrosine phosphatase SHP-1

The macrophage protein tyrosine phosphatase-1 SHP-1 has been implicated in the pathogenesis of infection with leishmania. To identify the factors that may interact with SHP-1, Leishmania donovani promastigote lysates were added to a GST-SHP-1 affinity matrix. A 44kDa specifically bound protein was identified as leishmania fructose-1,6-bisphosphate aldolase (aldolase). Purified leishmania aldolase bound to SHP-1 indicating that the interaction was direct. In contrast, purified mammalian aldolase did not bind to SHP-1. Consistent with this, leishmania aldolase activated SHP-1 in vitro, whereas mammalian aldolase did not. The presence of leishmania aldolase in the cytosolic fractions prepared from infected macrophages indicated that leishmania aldolase is exported from phagolysosomes in infected cells where it can target host cytosolic proteins. In fact, co-immunoprecipitation showed association of leishmania aldolase with SHP-1. Moreover, leishmania aldolase-expressing macrophages showed the deactivated phenotype of leishmania infected cells as judged by much reduced inability to induce expression of nitric-oxide synthase in response to interferon-γ treatment. Collectively, these data show that leishmania aldolase is a novel SHP-1 binding and activating protein that contributes to macrophage dysfunction.

Renal cortex neuronal nitric oxide synthase in response to rapamycin in kidney transplantation

Decreased renal neuronal nitric oxide synthase (nNOS) is present in various chronic kidney diseases although there is relative little known in chronic allograft nephropathy (CAN). Female sex increases the risk of acute rejection and calcineurin-inhibitor toxicity but decreases the risk of CAN. Rapamycin (RAPA) is an alternative immunosuppress although there is no information whether it is effective in females. We therefore investigated the efficacy of RAPA in both sexes and the impact of RAPA on renal cortex structure and nNOS expression. Male (M) and female (F) F344 kidneys were transplanted into same sex Lewis (ALLO) or F344 (ISO) recipients and treated with 1.6 mg/kg/day of RAPA for 10 days. Grafts were removed for renal histology and endothelial (e)NOS and neuronal (n)NOS protein measurements at 22 weeks. All ALLO rats survived without acute rejection. ALLO F survived with mild proteinuria and CAN at 22 weeks similar to ALLO M, while ISO F had better outcome than ISO M. Cortical nNOSα was undetectable in all RAPA groups; however, nNOSβ transcript and protein were compensatory increased. Both ALLO and ISO F showed higher medullary nNOSα but lower cortical eNOS abundance than M groups. In male ALLO RAPA decreased renal cortical nNOSα but increased nNOSβ expression. This may represent compensatory upregulation of nNOSβ when nNOSα-derived NO is deficient.

Expression of Nitric Oxide Synthase Isoforms in the Ovine Fetal Brain: Alteration by Hormonal and Hemodynamic Stimuli

Nitric oxide (NO) is synthesized in the brain through the action of three isoforms of nitric oxide synthase (NOS). The local generation of NO in neurons, glia, and vasculature modulates neuronal activity, as well as regional cerebral blood flow. We propose that, in the fetal brain, cerebral hypoperfusion alters the expression of NOS isoforms, and that estrogen administration modulates the NOS response to hypoperfusion. Sixteen chronically catheterized fetal sheep of known gestational age (124 to 128 days' gestation) were subjected to a 10-minute period of brachiocephalic occlusion (BCO) or to sham BCO; half of these fetuses were subjected to subcutaneous implant, which released 17beta-estradiol (E2; 0.25 mg/d) or placebo. Brain tissue was collected for mRNA and protein extraction 1 hour after the start of the BCO or sham BCO. All three isoforms of NOS were identified in fetal brain at both the mRNA and protein levels. BCO increased NOS1 (hippocampus, brainstem), NOS2 (hypothalamus), and NOS3 (hippocampus, cortex) at the protein level. Estradiol alone increased NOS1 (brainstem, cortex), NOS2 (hippocampus, hypothalamus), and NOS3 (brainstem, cerebellum) at the protein level, changes that were not mirrored at the mRNA level. The combination of BCO and estradiol produced smaller changes in NOS1 (brainstem, cortex), NOS2 (hippocampus, hypothalamus), and NOS3 (brainstem) protein levels than those produced by either stimulus alone. We conclude that the fetal brain expresses all isoforms of NOS, and that NOS expression is altered by both BCO and estradiol, but that the most prevalent effect of estradiol is to reduce specific NOS responses to cerebral hypoperfusion. The present results suggest the possibility that the neuroendocrine responses to estradiol and BCO are modulated by central nervous system (CNS) NO biosynthesis.

Role of neuronal nitric oxide synthase in response to hypertonic saline loading in rats

This study analyses the influence of neuronal nitric oxide synthase (nNOS) blockade with 7-nitroindazole (7NI) on the haemodynamic and renal response to a hypertonic saline load (HSL). We also evaluated the effects of non-specific NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). The following groups were used: controls, rats treated with 7NI at 0.5 or 5 mg kg(-1), and rats treated with L-NAME at 0.5 or 5 mg kg(-1). A further five groups received an isotonic saline load (ISL). Mean arterial pressure (MAP) was significantly increased in control rats after HSL. MAP was further increased in both 7NI-treated groups, and the L-NAME groups showed marked dose-related pressor responses. During ISL, MAP was only significantly increased in the group treated with 5 mg kg(-1) of L-NAME. The pressure-natriuresis relationship during the experimental period after the HSL was reduced in the 7NI group treated with 5 mg kg(-1) and severely attenuated in both L-NAME groups. The increase in plasma sodium was significantly greater after the HSL in both 7NI groups and both L-NAME groups compared with controls. The present results suggest that nNOS and other NOS isozymes play a counter-regulatory role in the pressor response to HSL. Moreover, the blockade of nNOS with the higher dose of 7NI produces a blunted pressure-natriuresis relationship in response to the HSL. Finally, it is concluded that nNOS participates in the homeostatic cardiovascular and renal response to hypertonic saline loading by attenuating the blood pressure increase and hypernatremia, and facilitating natriuresis.