Addition Of NO Donor Research Articles

Cell Type‐Specific Differential Effects of Nitric Oxide on PDE3 Expression in Pulmonary Vasculature

Persistent pulmonary hypertension of the newborn (PPHN) is an important cause of morbidity and mortality in neonates. Often used in combination with hyperoxia, the pulmonary vasodilator, inhaled nitric oxide (iNO), is the only FDA‐approved therapy for PPHN, although up to 40% of patients are non‐responders. In the pulmonary vasculature, NO increases cGMP levels, promoting vasodilation. cGMP competitively inhibits phosphodiesterase 3 (PDE3), thereby decreasing cAMP hydrolysis and further enhancing vasodilation. However, in animal models, NO treatment has been shown to increase PDE3 expression and/or activity and has been postulated to be the reason for rebound PH upon iNO withdrawal. Furthermore, it has been reported that that addition of a PDE3 inhibitor to iNO enhances pulmonary vasorelaxation. AMP‐activated protein kinase (AMPK), a critical regulator of energy homeostasis with downstream effects on NO synthase (NOS), has been shown to be regulated by PDE3 isoforms in other cell types and implicated in PH pathogenesis. We hypothesize that NO regulates the AMPK pathway via modulating PDE3 activity in a cell‐type specific manner in the pulmonary vasculature. Human PASMC (hPASMC) were treated with the NO donor DETA NONOate (1 mM) or DMSO and incubated in 21% or 85% O2 for 48 h. PDE3A, PDE3B, phosphorylated (p) and total (T)‐AMPK, p‐ and T‐ACC, and β‐actin protein expression were quantified by Western blot analyses. A similar experiment was performed using hPMVEC. Additionally, p‐ and T‐endothelial NOS (eNOS) protein expression were quantified. Our results show that hPASMC PDE3A protein expression was increased following Deta NONOate treatment in normoxia and hyperoxia (5.5±0.4 ‐fold, p=0.0004; 7.5±0.8 ‐fold, p<0.0001) relative to 21% O2 vehicle. Additionally, p/T‐AMPK protein expression was increased by 3‐fold after Deta NONOate treatment (p<0.0001). Consistent with these results, p/T‐ACC (a downstream target of AMPK), was also increased with the addition of NO donor in both conditions. In hPMVEC, the addition of NO donor increased PDE3A protein expression (2.4±0.1 ‐fold, p<0.0001). The combination of hyperoxia + NO donor, however, blunted the increase in PDE3A to a level similar to that in 21% O2, vehicle‐treated cells. Similarly, NO donor alone increased p/T‐AMPK (4.1±0.1 ‐fold, p<0.0001), whereas the combination of hyperoxia + NO blunted this increase. Moreover, p‐eNOS/β‐actin and T‐eNOS/β‐actin were both increased with the NO donor (1.8±0.03 ‐fold, p<0.0001; 2.9‐fold±0.20 SEM, p=0.0068), whereas the combination of hyperoxia + NO blunted the increase in p‐eNOS levels. In conclusion, exogenous NO donor increased PDE3A expression and AMPK activation in hPASMCs in both normoxia and hyperoxia. Alternatively, in hPMVEC, the increase in PDE3A expression, AMPK and eNOS activation after the addition of NO donor was blunted by hyperoxia. We speculate that these cell‐type specific differences in regulation of PDE3 and AMPK may be the reason for the variability in response of the pulmonary vasculature to PDE3 inhibition and AMPK modulators in PH.Support or Funding InformationNIH, NHLBI, R01, 1R01HL136963‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elevated mortality and prolonged intestinal cell death following combined radiation and burn in a mouse model

RESULTS: Endotoxemia results in increased hepatic antioxidant xpression as determined by PCR array. LPS-induced hepatic mitohondrial ROS both in vivo and in vitro. LPS also increased expresion of Nrf2. Addition of the NOS inhibitors L-NAME or 1400W ramatically reduced mitochondrial ROS production and comletely abrogated Nrf2 expression. This was associated with dereased expression of heme oxygenase-1 and superoxide dismutase-2. he addition of NO donor could rescue LPS-induced ROS producion and Nrf2 expression in the setting of NOS inhibition.

Proteomics investigation of endogenous S-nitrosylation in Arabidopsis

S-Nitrosylation emerges as an important protein modification in many processes. However, most data were obtained at the protein level after addition of a NO donor, particularly in plants where information about the cysteines nitrosylated in these proteins is scarce. An adapted work-flow, combining the classical biotin switch method and labeling with isotope-coded affinity tags (ICAT), is proposed. Without addition of NO donor, a total of 53 endogenous nitrosocysteines was identified in Arabidopsis cells, in proteins belonging to all cell territories, including membranes, and covering a large panel of functions. This first repertoire of nitrosothiols in plants enabled also preliminary structural description. Three apolar motifs, not located in close vicinity of cysteines and accounting for half the dataset, were detected and are proposed to complement nitrosylation prediction algorithms, poorly trained with plant data to date. Analysis of changes induced by a brief salt stress showed that NaCl modified the nitrosylation level of a small proportion of endogenously nitrosylated proteins and did not concern all nitrosothiols in these proteins. The possible role of some NO targets in the response to salt stress was discussed.

Nitric oxide‐mediated augmentation of neutrophil reactive oxygen and nitrogen species formation: Critical use of probes

Previous reports from this laboratory and others demonstrated NO-mediated biphasic modulation of NADPH oxidase and attenuation of neutrophil reactive oxygen species generation, whereas recently we reported augmentation in DCF fluorescence following NO treatment. These discrepancies seem to be due to utilization of different probes/methods to assess effect of NO on reactive oxygen and nitrogen species (ROS/RNS, reactive species) generation. This study aims to look into this and evaluate NO-mediated enzymatic reactive species formation by using multiple probes, human neutrophils/HL60 cells and various interventions. Addition of NO donor, SNP or SNAP (100 nM-1 mM) to PMNs suspension, exhibited a concentration- and time-dependent augmentation in DCF fluorescence, but reduced DHE fluorescence. Collective generation of reactive species was confirmed by enhanced DMPO-nitrone adduct, dityrosine and rhodamine-123 and quenching of scopoletin. NO also enhanced bacterial killing, without altering phagocytosis. Addition of NO to HL-60 cells lacking functional NADPH oxidase enhanced reactive species formation, indicating importance of other enzyme(s) too. NO-dependent ROS/RNS generation was substantially reduced by NADPH oxidase inhibitor (DPI), MPO inhibitor (ABAH), or NOS inhibitor (7-NI). However, 7-NI reduced MPO activity, warranting reappraisal of those reports, which implied NOS in reactive species formation. The results obtained demonstrated NO-mediated reactive species augmentation in human PMNs. Furthermore, superoxide scavenging by NO seems to be the key process in the decrease of DHE fluorescence and suggest usefulness of DCF as the most appropriate probe to measure the NO-mediated modulation of reactive oxygen species in particular in various pathological situations.

Berberine-induced apoptosis via decreasing the survivin protein in K562 cell line

Berberine is an isoquinoline alkaloid with multiple pharmacological activities, including anti-inflammatory and anti-diarrhea effect, the induction of apoptosis and anti-cancer effect. It has been reported that berberine exerts its anti-inflammatory effect via suppressing nuclear factor-kappa B (NF-κB) expression. Survivin and inducible nitric oxide synthase (iNOS) proteins may contribute to the causal relationship between anti-inflammatory and anti-apoptotic function. To investigate the mechanism of berberine-induced apoptotic activities, the human erythro-myeloblastoid leukemia cell line (K562 cell line) was treated with different concentrations of berberine (25-100μM). The most significant cellular growth arrest and apoptotic effects were observed in the cells treated with 75μM of berberine for 72h. The results indicate that survivin and iNOS protein levels were decreased in berberine-treated cells. However, decrease in the iNOS activity did not affect the cell growth and apoptosis. Moreover, the addition of NO donor, sodium nitroprusside, to culture medium decreased the cell growth in the present cell line, but it seemed that its concentration was too low to induce apoptosis. So despite its production by iNOS in untreated cells, NO does not play a significant role in carcinogenesis in this cell line. These results indicate that the apoptotic activity of berberine may be mediated through the reduction of survivin in K562 cells, but iNOS level and its activity does not play a significant role in berberine-induced apoptosis.

Opposite effects of nitric oxide on rod and cone photoreceptors of rat retina in situ

In the light-adapted vertebrate retina, nitric oxide (NO) modulates synaptic transmission between photoreceptors and second-order neurons. Although NO is believed to be a mediator of adaptation, its effect on photoreceptors in situ is not known yet. Therefore, we studied rod and cone activities in rat eyes in situ, using the electroretinogram (ERG). Rod and cone ERGs were functionally isolated by intravitreal 20 mM glutamate, which suppressed the activity of all retinal cells except rods and cones for about 90 min. The addition of NO-donor, SNAP, to the glutamate solution decreased the amplitude of the rod single-flash ERG by ∼40%, compared to the amplitude of the rod ERG isolated by glutamate alone, but it increased the amplitude of the isolated, intense paired-flash cone ERG by ∼40%. An excess of the NO-scavenger, CPTIO, had no significant effect on either rod or cone ERGs. A broad-spectrum NO-synthase inhibitor, L-NAME, increased the amplitude of the rod ERG by ∼50%, but had no significant effect on the cone ERG. We suggest that NO directly modulates the light-evoked activity of rod and cone photoreceptors in situ, but in opposite ways.

Formation of arteriovenous anastomosis during healing of acetic acid-induced ulcer: Inhibitory effect of bFGF and regulation by nNOS-positive nerve fibers

Considerable number of arteriovenous anastomoses (AVA) have been shown to exist in the ears and skin and to play an important role in blood flow regulation, while most studies concerning the gastric microcirculation have denied the existence of AVA. Aim: The present study was undertaken to clarify the alteration of the microcirculatory architecture during the healing of acetic acid-induced gastric ulcer, effect of basic fibroblast growth factor (bFGF) and changes of the autonomic nerve distribution. Methods: Wistar strain male rats, weighing 200 to 250 g, were used. Acetic acid-induced ulcers were induced by the direct application of 100% acetic acid on the serosal surface of the glandular stomach for 30 sec. Three, seven, and fourteen days after the treatment, a polyethylene catheter was inserted into either abdominal aorta or inferior vena cava and 4% aqueous solution of agar mixed with FITC-dextran was infused orthodromically or retrogradely, and fixed with Zamboni' s solution. 20J.L cryostat sections were made and observed by confocal laser microscopy (Zeiss LSM41O), which made it possible to reconstruct the microcirculatory system. In addition, some rats were treated with oral administration of CS23, acidstable human recombinant bFGF (IJ.LgllOOg b.w.) every twelve hours after the ulcer formation. To clarify the localization of cholinergic, adrenergic and neuronal nitric oxide synthase (nNOS)-positive nerve fibers, indirect fluorescent immunohistochemical studies were performed using anti-choline acetyltransferase, dopamine f3-hydroxylase and nNOS monoclonal antibodies. Results: In the control rat stomach, the gastric mucosal microvascular network consisted of arterioles, venules in the basal portion and true capillaries but no AVA were found. Fourteen days after ulcer formation, direct communication of arterioles and venules, i.e. AVA, was observed in the basal portion of the fundic mucosa in three out of five rats, while no AVA was found in the bFGF-treated rats. nNOS immunoreactive nerve fibers were densely distributed surrounding the AVA. The addition of NO donor, nitroprusside (40J.Lgl l OOg b.w.) brought about the marked opening of this AVA. Conclusions: From these observations, it was concluded that AVA was often recognized in the healing process of acetic acid-induced ulcer, while bFGF administration inhibited the AVA formation. This new blood channel was innervated by nNOS-positive nerve fibers and could be regulated by NO.