Increased neuronal nitric oxide synthase-derived NO production in the failing human heart

The pivotal role of neuronal nitric oxide synthase (nNOS) in regulating cardiac function has only recently been unveiled. Notably, others have shown that responsiveness to β-adrenergic stimulation is dependent on nNOS activity. In a cellular model, we showed that the Ca 2+ /calmodulin-dependent nNOS activity is reduced by overexpression of isoform 4b of the plasma membrane Ca 2+ /Calmodulin-dependent Ca 2+ -pump (PMCA4b), which binds to nNOS. We demonstrated that PMCA4b overexpression in the heart reduced β-adrenergic responsiveness in vivo via an nNOS dependent mechanism (Oceandy et al, Circulation 2007). Here we investigated the cellular mechanisms of the regulation of the β-adrenergic response by PMCA4b. We used an adenoviral system to overexpress PMCA4b (PMCA4b cells) or LacZ (control, C) in neonatal rat cardiomyocytes. PMCA4b cells showed an 18±5% and 24±5% reduction in nitric oxide (DAF-FM fluorescence) and cGMP levels, respectively (n=6, p<0.05 each) compared to C demonstrating the regulation of NO production by the PMCA4b in this system. Since nNOS has been shown to regulate phospholamban (PLB) phosphorylation, we examined phosphorylation of PLB at Ser16. PMCA4b cells showed a significant increase in Ser16-PLB at baseline (66±17%, p<0.05) compared to C. As a result of increased baseline Ser16-PLB in PMCA4b cells, β-adrenergic stimulation of PMCA4b cells using 2μM isoproter-enol (IP) showed reduced relative induction in Ser16-PLB (23±10% vs. 78±19% in C; n=5, p<0.05). Further analysis in adult cardiomyocytes isolated from our PMCA4b transgenic mice (PMCA4b TG) demonstrated that PMCA4b TG showed 3-fold higher Ser16-PLB phosphorylation at baseline compared to wild type (WT) myocytes and the relative response following β-adrenergic stimulation was significantly reduced (1.2±0.2 fold induction after IP treatment in PMCA4b TG, vs. 3.1±0.7 in WT, n=5, p<0.05). Thus, PMCA4b regulates NO production from nNOS, which in turn modulates cGMP levels and PLB phosphorylation. These findings provide mechanistic insight into the regulation of the β-adrenergic response in the heart by PMCA4b and place this Ca 2+ -pump upstream of the recently described pathway linking nNOS and Ser16-PLB phosphorylation and downstream of the β-adrenergic receptor(s).

Download Full-text

Neuronal nitric oxide synthase can mediate no production in mechanically stimulated mouse osteoblasts

Bone ◽

10.1016/j.bone.2011.03.400 ◽

2011 ◽

Vol 48 ◽

pp. S174

Author(s):

A.D. Bakker ◽

C. Huesa ◽

A. Hughes ◽

R.M. Aspden ◽

R.J. van 't Hof ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Neuronal Nitric Oxide Synthase ◽

No Production ◽

Oxide Synthase

Download Full-text

Neuronal nitric oxide synthase expression is lower in areas of the nucleus tractus solitarius excited by skeletal muscle reflexes in hypertensive rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00235.2012 ◽

2013 ◽

Vol 304 (11) ◽

pp. H1547-H1557 ◽

Cited By ~ 11

Author(s):

Megan N. Murphy ◽

Masaki Mizuno ◽

Ryan M. Downey ◽

John J. Squiers ◽

Kathryn E. Squiers ◽

...

Keyword(s):

Nitric Oxide ◽

Skeletal Muscle ◽

Nitric Oxide Synthase ◽

Protein Expression ◽

Nucleus Tractus Solitarius ◽

Neuronal Nitric Oxide Synthase ◽

No Production ◽

Hypertensive Rats ◽

Oxide Synthase ◽

Muscle Reflexes

The functions of the skeletal muscle exercise pressor reflex (EPR) and its mechanically sensitive component are augmented in hypertension producing exaggerated increases in blood pressure during exercise. Afferent information from the EPR is processed in the nucleus tractus solitarius (NTS). Within the NT, nitric oxide (NO), produced via l-arginine oxidation by neuronal nitric oxide synthase (nNOS), buffers the pressor response to EPR activation. Therefore, EPR overactivity may manifest as a decrease in NO production due to reductions in nNOS. We hypothesized that nNOS protein expression is lower in the NTS of spontaneously hypertensive (SHR) compared with normotensive Wistar-Kyoto (WKY) rats. Further, we examined whether nNOS is expressed with FOS, a marker of neuronal excitation induced by EPR activation. The EPR and mechanoreflex were intermittently activated for 1 h via hindlimb static contraction or stretch, respectively. These maneuvers produced significantly greater pressor responses in SHR during the first 25 min of stimulation. Within the NTS, nNOS expression was lower from −14.9 to −13.4 bregma in SHR compared with WKY. For example, at −14.5 bregma the number of NTS nNOS-positive cells in SHR (13 ± 1) was significantly less than WKY (23 ± 2). However, the number of FOS-positive cells after muscle contraction in this area was not different (WKY = 82 ± 18; SHR = 75 ± 8). In both groups, FOS-expressing neurons were located within the same areas of the NTS as neurons containing nNOS. These findings demonstrate that nNOS protein expression is lower within NTS areas excited by skeletal muscle reflexes in hypertensive rats.

Download Full-text

Subcellular localization of neuronal nitric oxide synthase in turtle retina: Electron immunocytochemistry

Visual Neuroscience ◽

10.1017/s0952523801186128 ◽

2001 ◽

Vol 18 (6) ◽

pp. 949-960 ◽

Cited By ~ 22

Author(s):

LUXIANG CAO ◽

WILLIAM D. ELDRED

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Bipolar Cell ◽

Plexiform Layer ◽

Neuronal Nitric Oxide Synthase ◽

No Production ◽

Inner Plexiform Layer ◽

Ribbon Synapses ◽

Cell Processes ◽

Oxide Synthase

Recent studies imaging nitric oxide (NO) production in the retina have indicated a much wider distribution of NO production than would be suggested by previous light-microscopic localizations of neuronal nitric oxide synthase (nNOS). To help resolve this discrepancy, the present study analyzed the ultrastructural localization of nNOS-like immunoreactivity (-LI) in all layers of the retina. In the ellipsoids of rod photoreceptors and the accessory elements of double cones, nNOS-LI was associated with some atypical mitochondria. In the outer plexiform layer, nNOS-LI was in some postsynaptic horizontal and bipolar cell processes at photoreceptor ribbon synapses. In some amacrine and ganglion cell somata, nNOS-LI was diffusely localized in the cytoplasm and associated with the endoplasmic reticulum. In the inner plexiform layer, nNOS-LI diffusely filled some amacrine cell processes, while in other amacrine cells nNOS-LI was selectively localized at the presynaptic specializations of conventional synapses. Neuronal NOS-LI was also found at membrane specializations in bipolar cell terminals that were distinct from their normal ribbon synapses. Finally, some nNOS-LI was found in mitochondria in Müller cells. The diverse subcellular localizations of nNOS-LI indicates that NO may play distinct functional roles in many retinal cells, which correlates well with the widespread NO production found in previous NO imaging studies.

Download Full-text

Defective Allele of the Neuronal Nitric Oxide Synthase Gene Increases Insulin Resistance During Acute Phase of Myocardial Infarction.

10.21203/rs.3.rs-285973/v1 ◽

2021 ◽

Author(s):

Otávio T Nóbrega ◽

Alessandra M. Campos-Staffico ◽

Elayne Kelen Oliveira ◽

Daniel B Munhoz ◽

Filipe A. Moura ◽

...

Keyword(s):

Nitric Oxide ◽

Myocardial Infarction ◽

Insulin Resistance ◽

Insulin Sensitivity ◽

Nitric Oxide Synthase ◽

Acute Phase ◽

Neuronal Nitric Oxide Synthase ◽

No Production ◽

Defective Allele ◽

Oxide Synthase

Abstract Background: glycemia disorders are a strong predictor of mortality in ST-Elevation Myocardial Infarction (STEMI) patients. Disruption in nitric oxide (NO) production is associated with insulin-resistant states. We evaluated whether NO production in carriers of a defective allele of the neuronal nitric oxide synthase (nNOS or NOS1), whose in vivo expression is reduced by up to 50%, might influence the insulin response during acute phase of STEMI. Methods and Results: Consecutive patients with STEMI (n = 354) underwent clinical evaluations and genotyping for the promoter variation rs41279104. Blood tests were performed at admission (D1) and after five days (D5) of in-hospital follow up, with the disposition index assessed in the period. Flow-mediated dilation (FMD) was assessed by reactive hyperemia on the 30th day. Homozygotes for the defective allele (A) showed lower glycemia and insulin sensitivity at D1 while showing the highest b-cells function and no changes in the circulating NO pool, what is compatible with hyperresponsive b-cells to counteract the inherent glucose-resistant state of AA patients. At D5, glycemic scores shifted to indicate greater insulin sensitivity among A homozygotes, paralleled by a slight yet poor increase in NO bioavailability than that among G carriers. All in all, defective homozygotes showed greater insulin resistance expressed by the disposition index at admission, which was compensated 5 days after STEMI even though FMD of A carriers was lower compared to G homozygotes. Conclusion: a defective nNOS allele seems to elicit endocrine adaptation and to associate with insulin resistance during the acute phase of STEMI.

Download Full-text

Downregulation of Neuronal Nitric Oxide Synthase in the Rat Remnant Kidney

Journal of the American Society of Nephrology ◽

10.1681/asn.v104704 ◽

1999 ◽

Vol 10 (4) ◽

pp. 704-713

Author(s):

AGNES ROCZNIAK ◽

JAMES N. FRYER ◽

DAVID Z. LEVINE ◽

KEVIN D. BURNS

Keyword(s):

Nitric Oxide ◽

Renal Failure ◽

Chronic Renal Failure ◽

Nitric Oxide Synthase ◽

Protein Expression ◽

Collecting Duct ◽

Neuronal Nitric Oxide Synthase ◽

No Production ◽

Oxide Synthase ◽

Nnos Expression

Abstract. Chronic renal failure is associated with disturbances in nitric oxide (NO) production. This study was conducted to determine the effect of 5/6 nephrectomy (5/6 Nx) on expression of intrarenal neuronal nitric oxide synthase (nNOS) in the rat. In normal rat kidney, nNOS protein was detected in the macula densa and in the cytoplasm and nuclei of cells of the inner medullary collecting duct by both immunofluorescence and electron microscopy. Western blot analysis revealed that 2 wk after 5/6 Nx, there were significant decreases in nNOS protein expression in renal cortex (sham: 95.42 ± 15.60 versus 5/6 Nx: 47.55 ± 12.78 arbitrary units, P < 0.05, n = 4) and inner medulla (sham: 147.70 ± 26.96 versus 5/6 Nx: 36.95 ± 17.24 arbitrary units, P < 0.005, n = 8). Losartan treatment was used to determine the role of angiotensin II (AngII) AT1 receptors in the inhibition of nNOS expression in 5/6 Nx. Losartan had no effect on the decreased expression of nNOS in the inner medulla, but partially increased nNOS protein expression in the cortex of 5/6 Nx rats. In contrast, in sham rats losartan significantly inhibited nNOS protein expression in the cortex (0.66 ± 0.04-fold of sham values, P < 0.05, n = 6) and inner medulla (0.74 ± 0.12-fold of sham values, P < 0.05, n = 6). nNOS mRNA was significantly decreased in cortex and inner medulla from 5/6 Nx rats, and the effects of losartan on nNOS mRNA paralleled those observed on nNOS protein expression. These data indicate that 5/6 Nx downregulates intrarenal nNOS mRNA and protein expression. In normal rats, AngII AT1 receptors exert a tonic stimulatory effect on expression of intrarenal nNOS. These findings suggest that the reduction in intrarenal nNOS expression in 5/6 Nx may play a role in contributing to hypertension and altered tubular transport responses in chronic renal failure.

Download Full-text

A Homogeneous Fluorescent Cell-Based Assay for Detection of Heterologously Expressed Nitric Oxide Synthase Activity

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105280640 ◽

2005 ◽

Vol 10 (8) ◽

pp. 849-855 ◽

Cited By ~ 5

Author(s):

Michael W. Wood ◽

Richard C. Hastings ◽

Linda A. Sygowski

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Neuronal Nitric Oxide Synthase ◽

No Production ◽

Fluorescent Cell ◽

Time Period ◽

Oxide Synthase ◽

Fold Stimulation ◽

Substrate Concentrations ◽

Nitric Oxide Synthase Activity

Arhodamine-derived, membrane-permeable fluorophore (DAR-4MAM) sensitive to nitric oxide production has been developed recently. The authors evaluated this reagent in both 96 and 384-well formats using heterologously expressed neuronal nitric oxide synthase (nNOS). nNOS transfected into HEK-293T cellswas stimulated by the addition of ionomycin. The calcium mobilization resulting from ionomycin treatment of nNOS-expressing 293T cells induced a robust increase in emission intensity, as measured using a standard rhodamine filter set. The effect was time dependent, and a 3 to 4-fold stimulation could be achieved in a 2-h time period. Ionomycin-dependent nitric oxide (NO) production was completely inhibited by several arginine analogs at micromolar concentrations (e.g., L-NAME IC 50= 3.0 µ M). Several arginine analog inhibitors of nNOS were revealed to be differentially reversible over increasing substrate concentrations. The assay is a facile method for characterizing inhibitors of nNOS in a relatively unperturbed cell environment.

Download Full-text

Potential roles of neuronal nitric oxide synthase and the PTEN-induced kinase 1 (PINK1)/Parkin pathway for mitochondrial protein degradation in disuse-induced soleus muscle atrophy in adult rats

PLoS ONE ◽

10.1371/journal.pone.0243660 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0243660

Author(s):

Munehiro Uda ◽

Toshinori Yoshihara ◽

Noriko Ichinoseki-Sekine ◽

Takeshi Baba ◽

Toshitada Yoshioka

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Protein Degradation ◽

Muscle Atrophy ◽

Soleus Muscle ◽

Neuronal Nitric Oxide Synthase ◽

Oxidative Modification ◽

No Production ◽

Adult Rats ◽

Oxide Synthase

Excessive nitric oxide (NO) production and mitochondrial dysfunction can activate protein degradation in disuse-induced skeletal muscle atrophy. However, the increase in NO production in atrophied muscles remains controversial. In addition, although several studies have investigated the PTEN-induced kinase 1 (PINK1)/Parkin pathway, a mitophagy pathway, in atrophied muscle, the involvement of this pathway in soleus muscle atrophy is unclear. In this study, we investigated the involvement of neuronal nitric oxide synthase (nNOS) and the PINK1/Parkin pathway in soleus muscle atrophy induced by 14 days of hindlimb unloading (HU) in adult rats. HU lowered the weight of the soleus muscles. nNOS expression showed an increase in atrophied soleus muscles. Although HU increased malondialdehyde as oxidative modification of the protein, it decreased 6-nitrotryptophan, a marker of protein nitration. Additionally, the nitrosocysteine content and S-nitrosylated Parkin were not altered, suggesting the absence of excessive nitrosative stress after HU. The expression of PINK1 and Parkin was also unchanged, whereas the expression of heat shock protein 70 (HSP70), which is required for Parkin activity, was reduced in atrophied soleus muscles. Moreover, we observed accumulation and reduced ubiquitination of high molecular weight mitofusin 2, which is a target of Parkin, in atrophied soleus muscles. These results indicate that excessive NO is not produced in atrophied soleus muscles despite nNOS accumulation, suggesting that excessive NO dose not mediate in soleus muscle atrophy at least after 14 days of HU. Furthermore, the PINK1/Parkin pathway may not play a role in mitophagy at this time point. In contrast, the activity of Parkin may be downregulated because of reduced HSP70 expression, which may contribute to attenuated degradation of target proteins in the atrophied soleus muscles after 14 days of HU. The present study provides new insights into the roles of nNOS and a protein degradation pathway in soleus muscle atrophy.

Download Full-text