Response of the Nitric Oxide System to Hypobaric Hypoxia in the Aged Striatum

Gerontology ◽  
2016 ◽  
Vol 63 (1) ◽  
pp. 36-44 ◽  
Author(s):  
Francisco Molina ◽  
María Luisa del Moral ◽  
María Ángeles Peinado ◽  
Alma Rus
Keyword(s):  
Nitric Oxide ◽  
Image Analysis ◽  
Hypobaric Hypoxia ◽  
Oxide System ◽  
Mrna Levels ◽  
Hypoxic Injury ◽  
Protein Levels ◽  
Enos Mrna ◽  
Nos Isoforms ◽  
Hypoxia Reoxygenation

Background: Nitric oxide (NO) appears to play a key role in the hypoxic injury to the brain. We have previously reported that hypoxia/reoxygenation downregulated NO synthases (NOS) in the adult striatum. Until now, no data were available concerning the influence of aging in conjunction with hypoxia/reoxygenation on the NO system in the striatum. Objective: The aim of this study was to assess the role of the NO pathway in the hypoxic aged striatum. Methods: Wistar rats 24-25 months old were submitted to hypobaric hypoxia (20 min)/reoxygenation (0 h, 24 h, 5 days). Expression (PCR, immunohistochemistry/image analysis) and activity (NADPH-diaphorase/image analysis) of NOS isoforms (neuronal NOS or nNOS, endothelial NOS or eNOS, inducible NOS or iNOS) were analyzed together with nitrated protein expression (immunohistochemistry/image analysis). NO levels were indirectly quantified as nitrates/nitrites (NOx). Results: The mRNA levels of NOS isoforms were undetectable at 0 h after hypoxia in the striatum compared to the control. At later reoxygenation times, nNOS mRNA decreased, while eNOS mRNA augmented. Protein levels of nNOS and eNOS rose at 24 h after hypoxia, and iNOS protein increased at 5 days. NOx levels remained unchanged, whereas in situ NOS activity and protein nitration diminished during reoxygenation in the aged striatum. Conclusion: The aged striatum may overexpress NOS isoforms as a neuroprotective-adaptive mechanism to hypoxia. However, this mechanism may not work properly in the aged striatum, since no changes in NO levels were detected after hypoxia. This may be related to the low activity of NOS isoforms in the hypoxic striatum.

Gene and protein expressions of nitric oxide synthases in ischemia-reperfused peripheral nerve of the rat

2001 ◽  
Vol 281 (3) ◽  
pp. C849-C856 ◽  
Author(s):  
Wen-Ning Qi ◽  
Zuo-Qin Yan ◽  
Peter G. Whang ◽  
Qi Zhou ◽  
Long-En Chen ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Peripheral Nerve ◽  
Ischemia Reperfusion ◽  
Nitric Oxide Synthases ◽  
Inos Mrna ◽  
Mrna Levels ◽  
Protein Expressions ◽  
Enos Mrna ◽  
Nos Isoforms ◽  
Endothelial Nitric Oxide

This study examined mRNA and protein expressions of neuronal (nNOS), inducible (iNOS), and endothelial nitric oxide synthases (eNOS) in peripheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. One sciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve were quantitatively measured by competitive PCR, and protein was determined by Western blotting and immunohistochemical staining. The results showed that, after ischemia (2 h), both nNOS and eNOS protein expressions decreased. After I/R (2 h of ischemia followed by 3 h of reperfusion), expression of both nNOS and eNOS mRNA and protein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal the dynamic expression of individual NOS isoforms during the course of I/R injury. An understanding of this modulation on a cellular and molecular level may lead to understanding the mechanisms of I/R injury and to methods of ameliorating peripheral nerve injury.

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

2002 ◽  
Vol 173 (2) ◽  
pp. 285-296 ◽  
Author(s):  
C Boiti ◽  
D Zampini ◽  
G Guelfi ◽  
F Paolocci ◽  
M Zerani ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Expression Patterns ◽  
Physiological Role ◽  
Total Activity ◽  
Pcr Analysis ◽  
Inos Mrna ◽  
Corpora Lutea ◽  
Mrna Levels ◽  
Isolated Cells ◽  
Protein Levels

Total activity of nitric oxide (NO) synthase (NOS) and expression of both endothelial (eNOS) and inducible (iNOS) isoforms were examined in corpora lutea (CL) of rabbits across pseudopregnancy by quantitative RT-PCR analysis, Western blot and immunohistochemistry. CL were collected at early- (day 4), mid- (day 9) and late- (day 13) luteal phases of pseudopregnancy. The PCR product of rabbit luteal eNOS was cloned and its direct sequence exhibited 90% homology with those of other species. The steady-state mRNA levels encoding eNOS remained fairly constant throughout both early- and mid-luteal stages of pseudopregnancy but dropped almost to half (P</=0.05) by day 13. By contrast, luteal eNOS proteins increased 2-fold (P</=0.05) from the early- to late-luteal phase. Independently of CL age, iNOS mRNA was very poorly expressed while protein levels gradually declined from the early- to late-luteal stage. Intense eNOS-like immunoreactivity was detected in large luteal cells, while iNOS staining was targeted to a few, isolated cells, probably macrophages. Basal NOS activity was greater in day 4 CL than in both day 9 and day 13 CL. These data are the first to characterize in rabbit CL the temporal expression patterns of NOS isoforms across different luteal stages of pseudopregnancy and, collectively, suggest the existence of an expressional control for this constitutive isoform, which might have a physiological role in regulating CL function during development.

scholarly journals Phytoestrogenic Effects of Blackcurrant Anthocyanins Increased Endothelial Nitric Oxide Synthase (eNOS) Expression in Human Endothelial Cells and Ovariectomized Rats

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1259 ◽  
Author(s):  
Kayo Horie ◽  
Naoki Nanashima ◽  
Hayato Maeda
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Endothelial Nitric Oxide Synthase ◽  
Mrna Levels ◽  
Human Endothelial Cells ◽  
Enos Expression ◽  
Ovx Rats ◽  
Enos Mrna ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

Phytoestrogens are plant-derived chemicals that are found in many foods and have estrogenic activity. We previously showed that blackcurrant extract (BCE) and anthocyanins have phytoestrogenic activity mediated via estrogen receptors (ERs), and anthocyanins may improve vascular function. BCE contains high levels of anthocyanins, but their health-promoting effects are unclear. This study examined the effects of BCE on the regulation of endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) synthesis in human endothelial cells as key regulators in cardiovascular disease. The results showed that eNOS mRNA levels were significantly upregulated in BCE- or anthocyanin-treated human vascular endothelial cells but decreased in cells treated with fulvestrant, an ER antagonist. These results corresponded with NO levels, suggesting that BCE and anthocyanin may regulate NO synthesis via eNOS expression. Thus, the phytoestrogenic effects exerted by BCE via ERs influenced eNOS mRNA expression and NO synthesis. In vivo, we investigated whether anthocyanin-rich BCE upregulated eNOS protein expression in ovariectomized (OVX) rats, a widely used animal model of menopause. Our results showed that anthocyanin-rich BCE significantly upregulated eNOS mRNA levels and NO synthesis through phytoestrogenic activity and therefore promoted blood vessel health in OVX rats as a postmenopausal model.

Enhanced gene expression of endothelial nitric oxide synthase in brown adipose tissue during cold exposure

2002 ◽  
Vol 282 (2) ◽  
pp. R623-R626 ◽  
Author(s):  
Kazue Kikuchi-Utsumi ◽  
Bihu Gao ◽  
Hiroshi Ohinata ◽  
Masaaki Hashimoto ◽  
Noriyuki Yamamoto ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Cold Exposure ◽  
Inos Mrna ◽  
No Production ◽  
Mrna Levels ◽  
Cold Stimulation ◽  
Brown Adipose ◽  
Enos Mrna

It has been shown that norepinephrine (NE) can mediate vasodilatation by stimulating the production of nitric oxide (NO) in brown adipose tissue (BAT), resulting in an increase in BAT blood flow. We speculated that constitutive NO synthase (NOS) is involved in this NO production. However, it is not known whether constitutive NOS is expressed in BAT. To answer this question, we assessed the expression of two types of constitutive NOS, endothelial (eNOS) and neuronal NOS (nNOS), in BAT of rats. eNOS was abundantly expressed in both BAT and isolated brown adipocytes, whereas nNOS was not. Cold exposure, which is known to stimulate NE release from sympathetic nerve terminals in BAT, led to a significant increase in eNOS mRNA in this tissue. In contrast, very low levels of inducible NOS (iNOS) mRNA were expressed, and cold stimulation failed to increase iNOS mRNA levels in BAT. These results suggest that eNOS is the primary isoform that is responsible for NO production in BAT and that its expression may be under sympathetic control.

Expression of nitric oxide synthase isoforms is reduced in late-gestation ovine fetal brainstem

2005 ◽  
Vol 289 (2) ◽  
pp. R613-R619 ◽  
Author(s):  
Charles E. Wood ◽  
Gin-Fu Chen ◽  
Maureen Keller-Wood
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Vascular Endothelial Cells ◽  
Late Gestation ◽  
Inos Mrna ◽  
Fetal Sheep ◽  
Enos Mrna ◽  
Ovine Fetal ◽  
Oxide Synthase ◽  
Nos Isoforms

Fetal baroreflex responsiveness increases in late gestation. An important modulator of baroreflex activity is the generation of nitric oxide in the brainstem nuclei that integrate afferent and efferent reflex activity. The present study was designed to test the hypothesis that nitric oxide synthase (NOS) isoforms are expressed in the fetal brainstem and that the expression of one or more of these enzymes is reduced in late gestation. Brainstem tissue was rapidly collected from fetal sheep of known gestational ages (80, 100, 120, 130, 145 days gestation and 1 day and 1 wk postnatal). Neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) mRNA was measured using real-time PCR methodology specific for ovine NOS isoforms. The three enzymes were measured at the protein level using Western blot methodology. In tissue prepared for histology separately, the cellular pattern of immunostaining was identified in medullae from late-gestation fetal sheep. Fetal brainstem contained mRNA and protein of all three NOS isoforms, with nNOS the most abundant, followed by iNOS and eNOS, respectively. nNOS and iNOS mRNA abundances were highest at 80 days' gestation, with statistically significant decreases in abundance in more mature fetuses and postnatal animals. nNOS and eNOS protein abundance also decreased as a function of developmental age. nNOS and eNOS were expressed in neurons, iNOS was expressed in glia, and eNOS was expressed in vascular endothelial cells. We conclude that all three isoforms of NOS are constitutively expressed within the fetal brainstem, and the expression of all three forms is reduced with advancing gestation. We speculate that the reduced expression of NOS in this brain region plays a role in the increased fetal baroreflex activity in late gestation.

Hypothalamic nitric oxide synthase is depressed in cholestatic rats

1997 ◽  
Vol 272 (5) ◽  
pp. G1034-G1040 ◽  
Author(s):  
M. G. Swain ◽  
T. Le ◽  
A. W. Tigley ◽  
P. Beck
Keyword(s):  
Nitric Oxide ◽  
Polymerase Chain Reaction ◽  
Steady State ◽  
Nitric Oxide Synthase ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Bile Duct Resection ◽  
Protein Levels ◽  
Polymerase Chain ◽  
Oxide Synthase

We examined hypothalamic nitric oxide synthase (NOS) levels and release as well as steady-state mRNA levels in rats with cholestasis due to bile duct resection (BDR) and in sham-resected control rats. BDR rats had a significant reduction in hypothalamic NOS-containing neurons in the hypothalamic paraventricular nucleus as determined by NADPH-diaphorase staining, compared with sham-resected controls. In addition, NOS activity, measured indirectly by determining nitrite release from hypothalamic explants, was significantly lower in BDR rats compared with sham-resected animals. Hypothalamic steady-state NOS mRNA levels [brain constitutive NOS (bNOS)] were determined by semiquantitative reverse transcription-polymerase chain reaction and were found to be increased 1.5-fold in BDR rats compared with sham rats. In summary, BDR rats have diminished hypothalamic NOS levels and activity coupled with enhanced steady-state bNOS mRNA levels, suggesting that depressed hypothalamic NOS protein levels are due to posttranscriptional defects.

Abstract 12692: Overexpression of Myocardial Inducible Nitric Oxide Synthase Exacerbates Cardiac Dysfunction and Beta-Adrenergic Desensitization in Streptozotocin-Induced Diabetic Mice

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Yixi Liu ◽  
Che Cheng ◽  
Xiaoqiang Sun ◽  
Jing Cao ◽  
Peng Zhou ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cardiac Dysfunction ◽  
Left Ventricular ◽  
Inos Expression ◽  
Diabetic Mice ◽  
Functional Responses ◽  
Protein Levels ◽  
Wild Type Female ◽  
Inos Inhibitor ◽  
Nos Isoforms

Background: Recent evidence links impaired nitric oxide (NO) signaling pathway in the pathogenesis of diabetes-induced cardiac dysfunction. However, the different nitric oxide synthases (NOS) isoforms involved in this pathology are controversial. Recent reports have shown that in failing myocardium, increased inducible NOS (iNOS) contributes to the attenuation of β-adrenergic receptor (AR)-mediated inotropic effect. The alteration and functional significance of cardiac iNOS in diabetic cardiomyopathy (DCM) are unclear. We assessed the hypothesis that increased cardiomyocyte iNOS expression and stimulation may inhibit myocyte contraction, relaxation, [Ca 2+ ] i transient ([Ca 2+ ] iT ), and depress its response to β-AR stimulation, thereby directly contributing to the functional impairment in DCM. Methods: Left ventricular (LV) myocyte protein levels of 3 NOS and myocyte functional responses were evaluated in 2 groups of wild-type female mice (11/group): control and DCM-induced by streptozotocin (STZ) (at 10 weeks after receiving 200 mg/kg STZ, ip). In DCM, we further assessed myocyte contractile and ([Ca 2+ ] iT ) responses to β-AR stimulation by isoproterenol (ISO, 10 -8 M) with and without pretreatment of myocytes with a selective iNOS inhibitor, 1400W (10 -5 M). Results: Compared with controls, DCM myocytes had significantly increased iNOS (1.76 vs 1.03) with decreased eNOS (0.29 vs 0.34), but relatively unchanged nNOS (0.17 vs 0.18). These changes were followed by significantly reduced basal cell contraction (dL/dt max , 73.8 vs 140.7 μm/s), relaxation (dR/dt max , 61.5 vs 121.1 μm/s) and [Ca 2+ ] iT (0.17 vs 0.20). ISO-stimulated increases in dL/dt max (32% vs 59%), dR/dt max (30% vs 55%) and [Ca 2+ ] iT (17% vs 31%) were also significantly reduced. Moreover, in DCM myocytes, pretreatment with 1400W markedly improved myocyte basal contraction (128.0 μm/s) and relaxation (111.7 μm/s). The ISO-induced increases in dL/dt max (55%), dR/dt max (52%), and [Ca 2+ ] iT (29%) were also significantly augmented. Conclusions: Our findings indicate that myocardial iNOS is activated in diabetic mice and suggest that increased iNOS expression contributes to depressed myocardial contractility, impaired [Ca 2+ ] i regulation and β-adrenergic hyporesponsiveness.

scholarly journals Chronic diet-induced hyperhomocysteinemia impairs eNOS regulation in mouse mesenteric arteries

2008 ◽  
Vol 295 (1) ◽  
pp. R59-R66 ◽  
Author(s):  
Robin C. Looft-Wilson ◽  
Blair S. Ashley ◽  
Janelle E. Billig ◽  
Madeline R. Wolfert ◽  
Lindsay A. Ambrecht ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Time Course ◽  
Ex Vivo ◽  
Microvascular Disease ◽  
Mesenteric Arteries ◽  
Mrna Levels ◽  
Enos Mrna ◽  
B Vitamin ◽  
Post Transcriptional Regulation ◽  
Endothelial Nitric Oxide

Hyperhomocysteinemia (HHcy) impairs endothelium-dependent vasodilation by increasing reactive oxygen species, thereby reducing nitric oxide (NO·) bioavailability. It is unclear whether reduced expression or function of the enzyme that produces NO·, endothelial nitric oxide synthase (eNOS), also contributes. It is also unclear whether resistance vessels that utilize both NO·and non-NO·vasodilatory mechanisms, undergo alteration of non-NO·mechanisms in this condition. We tested these hypotheses in male C57BL/6 mice with chronic HHcy induced by 6-wk high methionine/low-B vitamin feeding (Hcy: 89.2 ± 49.0 μM) compared with age-matched controls (Hcy: 6.6 ± 1.9 μM), using first-order mesenteric arteries. Dilation to ACh (10−9–10−4 M) was measured in isolated, cannulated, and pressurized (75 mmHg) arteries with and without N G-nitro-l-arginine methyl ester (l-NAME) (10−4 M) and/or indomethacin (10−5 M) to test endothelium-dependent dilation and non-NO·-dependent dilation, respectively. The time course of dilation to ACh (10−4 M) was examined to compare the initial transient dilation due to non-NO·, non-prostacyclin mechanism and the sustained dilation due to NO·. These experiments indicated that endothelium-dependent dilation was attenuated ( P < 0.05) in HHcy arteries due to downregulation of only NO·-dependent dilation. Western blot analysis indicated significantly less ( P < 0.05) basal eNOS and phospho-S1179-eNOS/eNOS in mesenteric arteries from HHcy mice but no difference in phospho-T495-eNOS/eNOS. S1179 eNOS phosphorylation was also significantly less in these arteries when stimulated with ACh ex vivo or in situ. Real-time PCR indicated no difference in eNOS mRNA levels. In conclusion, chronic diet-induced HHcy in mice impairs eNOS protein expression and phosphorylation at S1179, coincident with impaired NO·-dependent dilation, which implicates dysfunction in eNOS post-transcriptional regulation in the impaired endothelium-dependent vasodilation and microvascular disease that is common with HHcy.

Na-glucose and Na-neutral amino acid cotransport are uniquely regulated by constitutive nitric oxide in rabbit small intestinal villus cells

2005 ◽  
Vol 289 (6) ◽  
pp. G1030-G1035 ◽  
Author(s):  
Steven Coon ◽  
James Kim ◽  
Guohong Shao ◽  
Uma Sundaram
Keyword(s):  
Nitric Oxide ◽  
Small Intestine ◽  
Amino Acid ◽  
Basolateral Membrane ◽  
Kinetic Studies ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mechanism Of Inhibition ◽  
Rabbit Small Intestine ◽  
Villus Cell

Na-nutrient cotransport processes are not only important for the assimilation of essential nutrients but also for the absorption of Na in the mammalian small intestine. The effect of constitutive nitric oxide (cNO) on Na-glucose (SGLT-1) and Na-amino acid cotransport (NAcT) in the mammalian small intestine is unknown. Inhibition of cNO synthase with NG-nitro-l-arginine methyl ester (l-NAME) resulted in the inhibition of Na-stimulated 3H- O-methyl-d-glucose uptake in villus cells. However, Na-stimulated alanine uptake was not affected in these cells. The l-NAME-induced reduction in SGLT-1 in villus cells was not secondary to an alteration in basolateral membrane Na-K-ATPase activity, which provides the favorable Na gradient for this cotransport process. In fact, SGLT-1 was inhibited in villus cell brush-border membrane (BBM) vesicles prepared from animals treated with l-NAME. Kinetic studies demonstrated that the mechanism of inhibition of SGLT-1 was secondary to a decrease in the affinity for glucose without a change in the maximal rate of uptake of glucose. Northern blot studies demonstrated no change in the mRNA levels of SGLT-1. Western blot studies demonstrated no significant change in the immunoreactive protein levels of SGLT-1 in ileal villus cell BBM from l-NAME-treated rabbits. These studies indicate that inhibition of cNO production inhibits SGLT-1 but not NAcT in the rabbit small intestine. Therefore, whereas cNO promotes Na-glucose cotransport, it does not affect NAcT in the mammalian small intestine.

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