DETA NONOate Research Articles

Nitric Oxide Promotes Survival of Cerebral Cortex Neurons with Simultaneous Addition of [Fe(II)(β-Citryl-L-glutamate)] Complex in Primary Culture

It has been reported that the activity of mitochondrial aconitase (m-aconitase) is rapidly inhibited in a variety of cells when exposed to nitric oxide (NO). In present study, we found that NO significantly increased the number of surviving neurons via enhanced mitochondrial functions with simultaneous addition of the [Fe(II)(β-citryl-L-glutamate; β-CG)] complex. In vitro, a variety of aconitase-inhibitors, such as fluorocitrate, cyanide ion, ferricyanide ([Fe(CN)6]), and various oxidants including superoxide anion, inhibited the activity of m-aconitase even in the presence of Fe(II), whereas a NO-donor, nitroprusside (SNP) ([Fe(CN)5NO]), was the only agent that significantly increased activity of that enzyme. Therefore, it is reasonable to assume that NO released from SNP promotes Fe-dependent activation of aconitase. All other tested NO-donors, including 3-morpholino-sydnonimine (SIN), Deta NONOate (NOC18), and NaNO2, also promoted activation of m-aconitase in time- and dose-dependent manners in the presence of Fe(II). The promoting effects of the NO-donors on activation disappeared with the addition of NO-scavengers. In intact mitochondria, all tested NO-donors promoted reactivation of aconitase in a dose-dependent manner in the presence of Fe(II), whereas that was not seen in its absence. These findings suggest that NO released from NO-donors promotes Fe-dependent activation of aconitase. In mixed neuronal and glial cultures, NO-donors except for SNP enhanced mitochondrial activity at low concentrations. Furthermore, simultaneous addition of the [Fe(II)(β-CG)] complex significantly enhanced those activities and greatly increased the number of surviving neurons. Thus, NO can carry Fe ions into m-aconitase via the guide of the tag of β-CG addressed to the enzyme.

Alterations in the Circulating Nitrite Levels and Expression of NOS Isoforms in the Neutrophils of AML Patients

AbstractAbstract 4325 Introduction:Acute myeloid leukemia (AML) is a malignant hematopoietic neoplasm characterized by clonal proliferation of tumor cells that arise from the hematopoietic stem/progenitor population within the bone marrow. Nitric oxide (NO), a signaling molecule regulates hematopoietic stem and progenitor cells maturation by altering cell-cell adhesions, lineage-specific growth factors and actin confirmation (Michurina, et al, 2004). Moreover, suppression of NOS activity has profound effect on hematopoietic stem cells/progenitor cells, by increasing their self renewal or survival potential. Serum nitrite/nitrate level and expression of NOS is found to be more in AML patients (Brabdao et al, 2001). NO was also found to have cytotoxic potential in myeloid leukemia cells (Tsumori et al, 2002). Previous study from this lab has demonstrated that NO donor DETA NONOate in lower concentrations enhanced proliferation of HL-60 cells, while higher concentrations induced cytostasis, mitochondrial membrane potential loss and apoptosis (Kumar et al., 2010). Aim:The present study was undertaken to assess the circulating levels of NO metabolite, nitrite and NOS expressions in the circulating neutrophils and their precursor cells so as to assess the role of NO/NOS in AML pathology. Patients and Methods:Blood and bone marrow was collected from AML patients (mean age 33+10 years) after their informed consent. French American British criteria were used to diagnose AML pateints, which was based on the presence of 20% or more myeloblasts in bone marrow/blood smears. Twenty patients of AML were included and the study was approved by the Institutional ethics committee. Neutrophils (PMNs) and their precursor cells were isolated by Percoll density gradient and purity was confirmed by CD11b, CD15, CD16 labeling and by Giemsa staining. Total nitrite level in plasma and PMNs was assessed by Griess reagent and NOS expression was analysed by Real time RT-PCR. Results:Total nitrite level was less in AML patient's plasma and PMNs compared to healthy control (Figure 1A). Expression of both nNOS and iNOS was also less in AML patient's PMNs (Figure 1B). As AML is a disease of progenitor cells, further we have analysed nitrite level and NOS expression in PMNs progenitor cells {myeloblasts (MBs), promyelocytes (PMs), myelocytes (MCs), metamyelocytes (MMs), band cells (BCs) and segmented neutrophil (SNs)}. PMNs nitrite content and n/iNOS expression was less in all the progenitor cells compared to healthy control progenitor cells (Figure 2). In vitro results also showed that NOS expression was augmented following differentiation of HL60 cells in neutrophils. Conclusions:These observations indicate that NOS expression was significantly reduced in PMNs of AML patients. Further studies will be undertaken to assess the effect of NO modulators on the cell differentiation and apoptosis in AML patients. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Pendrin protein abundance in the kidney is regulated by nitric oxide and cAMP

Pendrin is a Cl(-)/HCO(3)(-) exchanger, expressed in the apical regions of some intercalated cell subtypes, and is critical in the pressor response to angiotensin II. Since angiotensin type 1 receptor inhibitors reduce renal pendrin protein abundance in mice in vivo through a mechanism that is dependent on nitric oxide (NO), we asked if NO modulates renal pendrin expression in vitro and explored the mechanism by which it occurs. Thus we quantified pendrin protein abundance by confocal fluorescent microscopy in cultured mouse cortical collecting ducts (CCDs) and connecting tubules (CNTs). After overnight culture, CCDs maintain their tubular structure and maintain a solute gradient when perfused in vitro. Pendrin protein abundance increased 67% in CNT and 53% in CCD when NO synthase was inhibited (N(G)-nitro-L-arginine methyl ester, 100 μM), while NO donor (DETA NONOate, 200 μM) application reduced pendrin protein by ∼33% in the CCD and CNT. When CNTs were cultured in the presence of the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (10 μM), NO donors did not alter pendrin abundance. Conversely, pendrin protein abundance rose when cAMP content was increased by the application of an adenylyl cyclase agonist (forskolin, 10 μM), a cAMP analog (8-bromo-cAMP, 1 mM), or a phosphodiesterase inhibitor (BAY60-7550, 50 μM). Since NO reduces cellular cAMP in the CNT, we asked if NO reduces pendrin abundance by reducing cAMP. With blockade of cGMP-stimulated phosphodiesterase II, NO did not alter pendrin protein abundance. We conclude that NO acts through cAMP to reduce pendrin total protein abundance by enhancing cAMP degradation.

Unique antineoplastic profile of Saquinavir-NO, a novel NO-derivative of the protease inhibitor Saquinavir, on the in vitro and in vivo tumor formation of A375 human melanoma cells

We have recently shown that covalent attachment of the nitric oxide (NO) moiety to the HIV protease inhibitor Saquinavir (Saq) produced a qualitatively new chemical entity, named Saquinavir-NO (Saq-NO), with enhanced anticancer properties and reduced toxicity both invitro and invivo. The aim of this study was to address several unanswered questions both on the pharmacological profile of Saq-NO as well as on the invivo role of NO in the oncogenesis of A375 human melanoma cells. To this end, we have evaluated here the impact of single and combined effects of Saq-NO, Saq, the NO-donor DETA NONOate and the iNOS inhibitor L-NAME on the invitro as well as invivo growth of the iNOS positive A375 cells. Our data confirm clear-cut evidence for a strong and powerful anti-melanoma action of Saq-NO that is not duplicable by the combined use of Saq and DETA NONOate. Surprisingly, but also in agreement with the complex and multifaceted role of endogenous NO in A375 cells, both DETA NONOate and L-NAME significantly suppressed the invivo growth of xenotransplants.

Nitric oxide-dependent killing of aerobic, anaerobic and persistent Burkholderia pseudomallei

Burkholderia pseudomallei infections are fastidious to treat with conventional antibiotic therapy, often involving a combination of drugs and long-term regimes. Bacterial genetic determinants contribute to the resistance of B. pseudomallei to many classes of antibiotics. In addition, anaerobiosis and hypoxia in abscesses typical of melioidosis select for persistent populations of B. pseudomallei refractory to a broad spectrum of antibacterials. We tested the susceptibility of B. pseudomallei to the drugs hydroxyurea, spermine NONOate and DETA NONOate that release nitric oxide (NO). Our investigations indicate that B. pseudomallei are killed by NO in a concentration and time-dependent fashion. The cytoxicity of this diatomic radical against B. pseudomallei depends on both the culture medium and growth phase of the bacteria. Rapidly growing, but not stationary phase, B. pseudomallei are readily killed upon exposure to the NO donor spermine NONOate. NO also has excellent antimicrobial activity against anaerobic B. pseudomallei. In addition, persistent bacteria highly resistant to most conventional antibiotics are remarkably susceptible to NO. Sublethal concentrations of NO inhibited the enzymatic activity of [4Fe–4S]-cofactored aconitase of aerobic and anaerobic B. pseudomallei. The strong anti-B. pseudomallei activity of NO described herein merits further studies on the application of NO-based antibiotics for the treatment of melioidosis.

EXOGENOUS NITRIC OXIDE (NO) MODULATES THE G‐PROTEIN COUPLED SIGNALING PROTEINS IN CULTURED LYMPHATIC SMOOTH MUSCLE CELLS

Lymphatics use both phasic and tonic contractions of lymphatic muscle. The cellular and molecular mechanisms by which lymphatic muscle performs these dual tasks are still unknown. Recently, we demonstrated that cGMP and PKG (protein kinase G) are involved in the NO modulation of rat thoracic duct contractility. We aim to study the NO dependent signaling mechanisms in cultured lymphatic muscle cells. Rat mesenteric lymphatic muscle cells (RMLMC) were used in this study. Cells were maintained in DMEM media supplemented with 10% fetal bovine serum and were incubated at 37°C and 5% CO2. Cells were treated with two NO donors SNAP (S‐Nitroso‐N‐Acetyl‐D,L‐Penicillamine) and DETA NONOate (Diethylenetriamine NONOate) at 10, 25, 50, 100 or 200 μM for 4 and 8 hours in different wells (n=6 in each treatment) separately. The sGC (soluble guanylate cyclase) isoforms α and β, PKG 1α, and PKG 1β were measured by Western blotting using anti‐sGC α, anti‐sGC β, anti‐PKG 1α, or anti‐PKG 1β antibodies. The expression of sGC α, sGC β, PKG 1α, and PKG 1β proteins were significantly upregulated in 25–200 μM SNAP and DETA NONOate treated RMLMC. Both dose and time dependent effects of SNAP and DETA NONOate on these proteins were observed. These findings demonstrate the usefulness of using cultured lymphatic muscle cell lines in the study of the involvement of sGC and PKG on NO dependent signaling. Supported by NIH HL70308

Impaired vasomotor function in uremic rats is directly related to NO‐resistance of smooth muscle cells

The number of patients with renal failure is 240 million worldwide, with 20% being dependent on dialysis. Renal failure increases the risk to develop cardiovascular diseases and could be related to vessel wall dysfunction.Gaining a better understanding of arterial dysfunction under uremic conditions and especially its effect on NO‐soluble guanylate cyclase (sGC) mediated vasorelaxation.For uremic rats, 5/6 nephrectomy (n=21) was performed in Wistar rats. After six weeks, both carotid arteries were removed and analyzed in a wire‐myograph. Different compounds were used to investigate NO‐dependent and ‐independent vasomotor pathways.SNP and Deta NONOate showed significant differences in vasorelaxation between sham and 5/6 nephrectomy group with a 0.5 log IC50 difference in NO‐induced relaxation (P<0.0001). This difference was endothelial independent as acetylcholine induced effects were blocked with L‐NAME. Also NO independent, but sGC heam dependent and independent, (Bay 41‐2272 and Bay 60‐2770, respectively) vasorelaxation showed significant differences (p<0.01 and p<0.0001). When sGC was oxidized with ODQ, sham group was more potent in relaxation for both compounds as compared to uremic rats.Reduced vasorelaxation in uremic rats is related to NO‐resistance of smooth muscle cells. Our results point toward an oxidized state of sGC as the main cause of vasomotor dysfunction in uremic rats.

Nitric oxide-releasing nanoparticles accelerate wound healing in NOD-SCID mice

Wound healing is a complex process, coordinated by various biological factors. In immunocompromised states wound healing can be interrupted as a result of decreased numbers of immune cells, impairing the production of effector molecules such as nitric oxide (NO). Therefore, topical NO-releasing platforms, such as diethylenetriamine (DETA NONOate), have been investigated to enhance wound healing. Recently, we demonstrated a nanoparticle platform that releases NO (NO-NPs) in a sustained manner, accelerating wound healing in both uninfected and infected murine wound models. Here, NO-NPs were investigated and compared to DETA NONOate in an immunocompromised wound model using non-obese, diabetic, severe combined immunodeficiency mice. NO-NP treatment accelerated wound closure as compared to controls and DETA NONOate treatment. In addition, histological assessment revealed that wounds treated with NO-NPs had less inflammation, more collagen deposition, and more blood vessel formation as compared to other groups, consistent with our previous data in immunocompetent animals. These data suggest that NO-NPs may serve as a novel wound-healing therapy in the setting of immunocompromised states associated with impaired wound healing. From the Clinical EditorWound healing in an immunocompromised host is often incomplete and is a source of major concern in such conditions. This work demonstrates in a murine model that in these settings NO releasing nanoparticles significantly enhance wound healing.

Advanced polymeric matrix for valvular complications

Poly(L-lactic acid) (PLLA) matrix systems incorporated with poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) containing nitric oxide (NO) donors (DETA NONOate) were developed for prevention of heart valve complications through sustained and controlled release of NO. PLLA matrices were prepared using the salt leaching method and the properties and drug release profiles were characterized. For assessment of the effects of PLLA systems on the pharmacological responses and cytotoxicity, various factors, such as calcium content, alkaline phosphatase (ALP) activity, cyclic guanosine monophosphate (cGMP) expression, intercellular adhesion molecule (ICAM-1) expression and cell viability of porcine aortic valve interstitial cells (PAVICs), were evaluated. PLLA matrices embedded with PLGA- NPs demonstrated its usefulness in alleviating the calcification rate of the VICs. The cGMP levels under osteoblastic conditions significantly increased, supporting that anticalcification activity of NO is mediated through NO-cGMP signaling pathway. The level of ICAM-1 expression in cells exposed to NO was lowered, suggesting that NO has an inhibitory activity against tissue inflammation. NO releases from PLLA matrix embedded with PLGA NPs prevented valvular calcification and inflammation without causing any cytotoxic activities. PLLA matrix system loaded with NPs containing NO donors could provide a new platform for sustained and controlled delivery of NO, significantly reducing valvular complications.

Abstract 3063: Nitroglycerin Treatment Attenuates APP and BACE1 Protein Levels In Cerebral Microcirculation

Several cardiovascular risk factors are associated with an increased incidence of Alzheimer’s disease (AD); however, the mechanistic link is unknown. A common feature of cardiovascular risk factors is endothelial dysfunction, specifically, loss of bioavailability of endothelial nitric oxide (NO). Recently, we demonstrated that loss of endothelial NO led to increased protein levels of amyloid precursor protein (APP), β-site APP cleaving enzyme 1 (BACE1) and beta amyloid (Aβ) peptide. APP, the parent protein, when sequentially cleaved by BACE1 and γ-secretase generates amyloidogenic, Aβ, the primary component of AD plaques. Our data was the first to demonstrate a possible mechanism linking endothelial dysfunction, via loss of endothelial NO, with the upregulation of important AD related proteins in the cerebral microvasculature. Therefore, we sought to determine if supplementation of cultured human brain microvascular endothelial cells with NO, by utilizing the NO donor, DETA NONOate, would attenuate APP and BACE1 protein. DETA NONOate, 10 -8 to 10 -6 M, reduced expression of APP and BACE1 as compared to untreated control cells (n=3). To test the effects of NO supplementation in vivo, we treated endothelial nitric oxide synthase deficient (eNOS -/- ) mice with 30 mg/kg nitroglycerine b.i.d. or vehicle via subcutaneous injections for 3 days. No difference was observed in blood pressure between animals treated with vehicle (n=7) or nitroglycerine (n=6) (P>0.05) or before and after nitroglycerine treatment (n=6, P>0.05). As expected, cerebral microvessles of eNOS -/- animals treated with nitroglycerine displayed increased levels of cGMP as compared to vehicle (n=7-8; P<0.05). Moreover, APP and BACE1 protein levels were statistically lower in cerebral microvessels from eNOS -/- mice treated with nitroglycerine as compared to vehicle treated (n=6-7, APP, P<0.05; BACE1, P<0.05). These data indicate that supplementation with NO is able to suppress APP and BACE1 levels in the cerebrovascular endothelium. Our findings suggest that preservation of NO/cGMP signaling may be an important therapeutic target in preventing and treating mild cognitive impairment as well as AD.

Prolonged nitric oxide treatment induces tau aggregation in SH-SY5Y cells

Presence of cytoplasmic tau aggregates is a hallmark of brains in patients with tauopathies such as Alzheimer's disease. However, the mechanism underlying formation of these insoluble tau aggregates remains elusive. In this study, we investigated the impact of prolonged nitric oxide (NO) exposure on neuronal SH-SY5Y cells overexpressing human tau. Treatment with the NO donor DETA NONOate for up to 48h resulted in an increase in S-nitrosation of cellular proteins, inactivation of proteasome, and impairment of respiration. Western blot analysis of Triton X-soluble fractions of NO-treated cells revealed that persistent NO treatment increased heterogeneity in tau molecule size, as a result of dephosphorylation, and induced the formation of sodium dodecyl sulfate (SDS)-stable oligomeric tau aggregates, stabilized by disulfide bonds. Moreover, further NO treatment induced the formation of SDS-stable insoluble tau mega-aggregates that were composed of dephosphorylated full-length tau molecules and other proteins, and were stabilized through disulfide bonds. Evaluation of the role of these tau aggregates as potential seeds for tau fibrillization and elucidation of their formation mechanism in our model, could lead to better understanding of the pathogenesis of tauopathies.

Exogenous, but not Endogenous Nitric Oxide Inhibits Adhesion Molecule Expression in Human Endothelial Cells

Nitric oxide (NO) has many beneficial actions on the vascular wall including suppression of inflammation. The mechanism(s) by which NO antagonizes cytokine signaling are poorly understood, but are thought to involve inhibition of the pro-inflammatory transcription factor, NF-κB. NO represses nuclear translocation of NF-κB via the S-nitrosylation of its subunits which decreases the expression of target genes including adhesion molecules. In previous studies, we have shown that the intracellular location of endothelial nitric oxide synthase (eNOS) can influence the amount of NO produced and that NO levels are paramount in regulating the S-nitrosylation of target proteins. The purpose of the current study was to investigate the significance of subcellular eNOS to NF-κB signaling induced by pro-inflammatory cytokines in human aortic endothelial cells (HAECs). We found that in HAECs stimulated with TNFα, L-NAME did not influence the expression of intercellular adhesion molecule 1 (ICAM-1) or vascular cell adhesion molecular 1 (VCAM-1). In eNOS “knock down” HAECs reconstituted with either plasma membrane or Golgi restricted forms of eNOS, there was no significant effect on the activation of the NF-κB pathway over different times and concentrations of TNFα. Similarly, the endogenous production of NO did not influence the phosphorylation of IκBα. In contrast, higher concentrations of NO derived from the use of the exogenous NO donor, DETA NONOate, effectively suppressed the expression of ICAM-1/VCAM-1 in response to TNFα and induced greater S-nitrosylation of IKKβ and p65. Collectively these results suggest that neither endogenous eNOS nor eNOS location is an important influence on inflammatory signaling via the NF-κB pathway and that higher NO concentrations are required to suppress NF-κB in HAECs.

Characterization of diazeniumdiolate nitric oxide donors (NONOates) by electrospray ionization mass spectrometry

Diazeniumdiolates (also called NONOates) have been analyzed by electrospray ionization mass spectrometry (ESI-MS). The samples used are commercially available and included Diethylamine NONOate, DETA NONOate, Spermine NONOate, MAHMA NONOate, PROLI NONOate, Dipropylenetriamine NONOate, PAPA NONOate, and Sulpho NONOate. These compounds have been found to ionize upon ESI by protonation, deprotonation and sodiation. The MS(n) experiments provided strong evidence that such ions release NO, HNO, N(2)O, NO(2), N(2)O(2), N(3)O(3), N(4)O(3) and N(4)O(4) when collisionally activated. Thus, the facile donation of NO units is a property of such compounds. Negative-mode mass spectrometry has been particularly useful for the analysis of most of the NONOates studied here. The experiments have demonstrated the capabilities of mass spectrometry, along with CAD (MS/MS), to detect and characterize such compounds.

Exogenous intravascular nitric oxide enhances ventricular function after hemodilution with plasma expander

Aims This study evaluated the hypothesis that exogenous nitric oxide (NO) supplementation during acute hemodilution with plasma expander (PE) provides beneficial effects on cardiac function. Main methods Acute hemodilution in golden Syrian hamsters was induced by a 40% of blood volume exchange with dextran 70 kDa. Intravascular NO supplementation after hemodilution was accomplished with a NO donor, diethylenetriamine NONOate (DETA NONOate). The test group was treated with DETA NONOate, while the control group received only vehicle. Left ventricular cardiac function was studied using pressure–volume measurements obtained with a miniaturized conductance catheter. Key findings Cardiac output increased to 122 ± 5% and 107 ± 1% of the baseline in the group treated with NO donor and the vehicle group, respectively. Stroke work per stroke volume (SW/SV) after hemodilution reduced to 90% of the baseline and the NO donor significantly reduced SW/SV compared to the vehicle. The minimum rate of pressure change (dP/dt min) was significantly lower in animals treated with the NO donor compared to vehicle treated animals. Systemic vascular resistance (SVR) decreased to 62 ± 5% of the baseline in the NO donor group whereas the vehicle group SVR decreased to 83 ± 5% of the baseline. Using intravital microscopy analysis of microvessel in the dorsal skinfold window chamber, we established that the NO donor group induced significant vasodilation compared to the vehicle group. Significance NO supplementation in an acute hemodilution with PE has beneficial effects on cardiac performance. However, the NO supplementation effects with a NO donor are dose-independent and short-lasting.

Heterogeneity in relaxation of different sized porcine coronary arteries to nitrovasodilators: role of PKG and MYPT1

The present study was to determine the role of the type I isoform of cGMP-dependent protein kinase (PKG I) and its downstream effector myosin phosphatase target subunit 1 (MYPT1) in the responses of different sized coronary arteries to nitrovasodilators. Relaxations of isolated porcine coronary arteries were determined by isometric tension recording technique. Protein levels of PKG I and its effectors were analyzed by Western blotting. The activities of PKG I and MYPT1 were studied by analyzing phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and MYPT1, respectively. Nitroglycerin, DETA NONOate, and 8-Br-cGMP caused greater relaxations in large than in small coronary arteries. Relaxations were attenuated to a greater extent by Rp-8-Br-PET-cGMPS (a PKG inhibitor) in large vs. small arteries. The expressions of PKG I and MYPT1 in large arteries were more abundant than in small arteries. DETA NONOate stimulated phosphorylation of VASP at Ser239 and inhibited phosphorylation of MYPT1 at Thr853 to a greater extent in large than in small arteries. A suppressed phosphorylation of MYPT1 at Thr853 was caused by 8-Br-cGMP in large but not small arteries, which was inhibited by Rp-8-Br-PET-cGMPS. These results suggest that the greater responsiveness of large coronary arteries to nitrovasodilators result in part from greater activities of PKG I and MYPT1. Dysfunction in nitric oxide signaling is implicated in the vulnerability of large coronary arteries to certain disorders such as atherosclerosis and spasm. Augmentation of PKG I-MYPT1 signaling may be of therapeutic benefit for combating these events.

Endothelium-derived NO, but not cyclic GMP, is required for hypoxic augmentation in isolated porcine coronary arteries

The present study investigated the mechanism underlying the transient potentiation of vasoconstriction by hypoxia in isolated porcine coronary arteries. Isometric tension was measured in rings with or without endothelium. Hypoxia (Po(2) <30 mmHg) caused a transient further increase in tension (hypoxic augmentation) in contracted (with U46619) preparations. The hypoxic response was endothelium dependent and abolished by inhibitors of nitric oxide synthase [N(ω)-nitro-L-arginine methyl ester (L-NAME)] or soluble guanylyl cyclase (ODQ and NS2028). The addition of DETA NONOate (nitric oxide donor) in the presence of L-NAME restored the hypoxic augmentation, suggesting the involvement of the nitric oxide pathway. However, the same was not observed after incubation with 8-bromo-cyclic GMP, atrial natriuretic peptide, or isoproterenol. Assay of the cyclic GMP content showed no change upon exposure to hypoxia in preparations with and without endothelium. Incubation with protein kinase G and protein kinase A inhibitors did not inhibit the hypoxic augmentation. Thus the hypoxic augmentation is dependent on nitric oxide and soluble guanylyl cyclase but independent of cyclic GMP. The hypoxic augmentation persisted in calcium-free buffer and in the presence of nifedipine, ruling out a role for extracellular calcium influx. Hypoxia did not alter the intracellular calcium concentration, as measured by confocal fluorescence microscopy. This observation and the findings that hypoxic augmentation is enhanced by thapsigargin (sarco/endoplasmic reticulum calcium ATPase inhibitor) and inhibited by HA1077 or Y27632 (Rho kinase inhibitors) demonstrate the involvement of calcium sensitization in the phenomenon.

Exposure to nitric oxide increases the nitrosyl-iron complexes content in sorghum embryonic axes

This work was aimed to investigate nitrosyl-Fe complexes formation by reaction of endogenous ligands and Fe, in sorghum embryonic axes exposed to NO-donors. Electron paramagnetic resonance (EPR) was employed to detect the presence of nitrosyl-Fe complexes in plant embryos, as well as changes in labile iron pool (LIP). Nitrosyl-Fe complexes formation was detected in sorghum embryonic axes homogenates incubated in vitro in the presence of 1 mM of NO donors: diethylenetriamine NONOate (DETA NONOate), S-nitrosoglutathione (GSNO) and sodium nitroprusside (SNP). In axes isolated from seeds incubated in vivo in the presence of 1 mM SNP for 24 h, the content of NO was increased by 2-fold, and the EPR spectrum from mononitrosyl-Fe complexes (MNIC) was observed with a concomitant increase in the fresh weight of sorghum axes. The simultaneous exposure to deferoxamine and the NO donor precluded the increase in fresh weight observed in the presence of excess NO. While total Fe content in the axes isolated from seeds exposed to 1 mM SNP was not significantly affected as compared to control axes, the LIP was increased by over 2-fold.The data reported suggest a critical role for the generation of complexes between Fe and NO when cells faced a situation leading to a significant increase in NO content. Moreover, demonstrate the presence of MNICs as one of the important components of the LIP, which could actively participate in Fe cellular mobilization.

Distinct role of nitric oxide and peroxynitrite in mediating oligodendrocyte toxicity in culture and in experimental autoimmune encephalomyelitis

Nitric oxide has been implicated in the pathogenesis of multiple sclerosis. However, it is still unclear whether nitric oxide plays a protective role or is deleterious. We have previously shown that peroxynitrite, a reaction product of nitric oxide and superoxide, is toxic to mature oligodendrocytes (OLs). The toxicity is mediated by intracellular zinc release, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), activation of 12-lipoxygenase (12-LOX) and the formation of reactive oxygen species (ROS). In this study, we found that the donors of nitric oxide, dipropylenetriamine NONOate (DPT NONOate) and diethylenetriamine NONOate (DETA NONOate), protected OLs from peroxynitrite or zinc-induced toxicity. The protective mechanisms appear to be attributable to their inhibition of peroxynitrite- or zinc-induced ERK1/2 phosphorylation and 12-LOX activation. In cultures of mature OLs exposed to lipopolysaccharide (LPS), induction of inducible nitric oxide synthase (iNOS) generated nitric oxide and rendered OLs resistant to peroxynitrite-induced toxicity. The protection was eliminated when 1400W, a specific inhibitor of iNOS, was co-applied with LPS. Using MOG35-55-induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, we found that nitrotyrosine immunoreactivity, an indicator of peroxynitrite formation, was increased in the spinal cord white matter, which correlated with the loss of mature OLs. Targeted gene deletion of the NADPH oxidase component gp91phox reduced clinical scores, the formation of nitrotyrosine and the loss of mature OLs. These results suggest that blocking the formation specifically of peroxynitrite, rather than nitric oxide, may be a protective strategy against oxidative stress induced toxicity to OLs.

Nitric oxide reduces renal pendrin abundance

Pendrin is an angiotensin II-sensitive intercalated cell Cl− transporter that modulates blood pressure. We showed that angiotensin type 1 receptor inhibitors reduce renal pendrin total protein abundance in mice in vivo through a nitric oxide (NO)-dependent mechanism. We used western blots and immunohistochemistry to determine if NO modulates renal pendrin expression in vivo. Nitric oxide synthase inhibitor (L-NAME, 0.4 mg/d) administration for 7 d increased pendrin total protein abundance ~25%, without changing its subcellular distribution. To study pendrin's regulation in vitro, we dissected and cultured mouse cortical collecting ducts (CCDs) and connecting tubules (CNTs) up to 18 hrs. After 18 hrs of culture, CCDs maintain a tubule structure and a steep solute gradient when perfused in vitro. Thus, we quantified pendrin protein abundance by confocal fluorescent microscopy in tubules from furosemide-treated mice after 0–18 hrs of culture. Pendrin protein abundance is relatively stable in untreated CCDs and CNTs during 18 hrs of culture but increases 67% in CNT and 53% in CCD with 18 hrs of L-NAME administration (100 μM). Conversely, NO donor (DETA NONOate, 200 μM) application (18 hrs) reduces pendrin protein in CCD 36%. Thus, NO reduces pendrin protein abundance in CNT and CCD in vivo and in vitro. The signaling mechanism by which this occurs remains to be determined.

INHIBITION OF NITRIC OXIDE SYNTHASE ENHANCES THE PRODUCTION OF TUMOR NECROSIS FACTOR ‐ALPHA IN MACROPHAGE CELLS

We demonstrated earlier that systemic infusion of nitric oxide (NO) synthase inhibitor, nitro-L-arginine methyl ester (L-NAME) caused an increase in the plasma level of tumor necrosis factor-alpha (TNF-α) in mice. However, the source of TNF-α release during systemic NO blockade is not known. To determine the source, cultured macrophage cells were treated with L-NAME (100 μM; Sigma) or vehicle, and were incubated for 1, 2, 4 and 6 hours (n=6 in each treatment). TNF-α concentration in the condition media, harvested after each incubation period, was measured using a quantitative TNF-α ELISA kit. In another set, L-NAME treated cells were co-treated with a NO donor compound, Diethylenetriaamine NONOate, (DETA NONOate; 1 mM). The mRNA levels of T TNF-α was measured in cell extract by real time RT-PCR. TNF-α level in the condition media was increased in a time dependent manner in cells treated with L-NAME compared to vehicle (316 ± 68 vs 143 ± 32 pg/mL in 1st hour and 1459 ± 348 vs 414 ± 82 pg/mL in 6th hour). Compared to vehicle, there were also increases in TNF-α mRNA (0.62 ± 0.02 vs 1.68 ± 0.05 relative folds changes at 6thhour period) in cells treated with L-NAME. Co-treatment with L-NAME and DETA NONOate prevented the increased TNF-α production/release as well as mRNA expression of TNF-α. These data demonstrate that NO production exerts a tonic inhibition on TNF-α production and release in macrophage cells.