MAHMA NONOate Research Articles

Exploitation of nitric oxide donors to control bacterial adhesion on ready-to-eat vegetables and dispersal of pathogenic biofilm from polypropylene.

Nitric oxide (NO) donors have been used to control biofilm formation. Nitric oxide can be delivered in situ using organic carriers and acts as a signaling molecule. Cells exposed to NO shift from biofilm to the planktonic state and are better exposed to the action of disinfectants. In this study, we investigate the capability of the NO donors molsidomine, MAHAMA NONOate, NO-aspirin and diethylamine NONOate to act as anti-adhesion agents on ready-to-eat vegetables, as well as dispersants for a number of pathogenic biofilms on plastic. Our results showed that 10 pM molsidomine reduced the attachment of Salmonella enterica sv Typhimurium 14 028 to pea shoots and coriander leaves of about 0.5 Log(CFU/leaf) when compared with untreated control. The association of 10 pmol L-1 molsidomine with 0.006% H2 O2 showed a synergistic effect, leading to a significant reduction in cell collection on the surface of the vegetable of about 1 Log(CFU/leaf). Similar results were obtained for MAHMA NONOate. We also showed that the association of diethylamine NONOate at 10 mmol L-1 and 10 pmol L-1 with the quaternary ammonium compound diquat bromide improved the effectiveness of biofilm dispersal by 50% when compared with the donor alone. Our findings reveal a dual role of NO compounds in biofilm control. Molsidomine, MAHMA NONOate, and diethylamine NONOate are good candidates for either preventing biofilm formation or dispersing biofilm, especially when used in conjunction with disinfectants. Nitric oxide compounds have the potential to be developed into a toolkit for pro-active practices for good agricultural practices (GAPs), hazard analysis and critical control points (HACCP), and cleaning-in-place (CIP) protocols in industrial settings where washing is routinely applied. © 2020 Society of Chemical Industry.

Detection of Nitric Oxide by Membrane Inlet Mass Spectrometry.

Membrane inlet mass spectrometry (MIMS) is a reproducible and reliable method for the measurement of nitric oxide in aqueous solution with a lower limit of detection of 10nM and a linear response to 50μM. MIMS utilizes a semipermeable membrane to partition analytes based on physicochemical properties from the bulk sample into the mass spectrometer. Silastic tubing allows the introduction of small gaseous molecules including nitric oxide (NO) into the high vacuum of a mass spectrometer. We describe the measurement of NO generated chemically from nitrite and MAHMA NONOate as well as enzymatically by nitric oxide synthase (NOS).

Physiological Levels of Nitric Oxide Diminish Mitochondrial Superoxide. Potential Role of Mitochondrial Dinitrosyl Iron Complexes and Nitrosothiols.

Mitochondria are the major source of superoxide radicals and superoxide overproduction contributes to cardiovascular diseases and metabolic disorders. Endothelial dysfunction and diminished nitric oxide levels are early steps in the development of these pathological conditions. It is known that physiological production of nitric oxide reduces oxidative stress and inflammation, however, the precise mechanism of “antioxidant” effect of nitric oxide is not clear. In this work we tested the hypothesis that physiological levels of nitric oxide diminish mitochondrial superoxide production without inhibition of mitochondrial respiration. In order to test this hypothesis we analyzed effect of low physiological fluxes of nitric oxide (20 nM/min) on superoxide and hydrogen peroxide production by ESR spin probes and Amplex Red in isolated rat brain mitochondria. Indeed, low levels of nitric oxide substantially attenuated both basal and antimycin A-stimulated production of reactive oxygen species in the presence of succinate or glutamate/malate as mitochondrial substrates. Furthermore, slow releasing NO donor DPTA-NONOate (100 μM) did not change oxygen consumption in State 4 and State 3. However, the NO-donor strongly inhibited oxygen consumption in the presence of uncoupling agent CCCP, which is likely associated with inhibition of the over-reduced complex IV in uncoupled mitochondria. We have examined accumulation of dinitrosyl iron complexes and nitrosothiols in mitochondria treated with fast-releasing NO donor MAHMA NONOate (10 μM) for 30 min until complete release of NO. Following treatment with NO donor, mitochondria were frozen for direct detection of dinitrosyl iron complexes using Electron Spin Resonance (ESR) while accumulation of nitrosothiols was measured by ferrous-N-Methyl-D-glucamine dithiocarbamate complex, Fe(MGD)2, in lysed mitochondria. Treatment of mitochondria with NO-donor gave rise to ESR signal of dinitrosyl iron complexes while ESR spectra of Fe(MGD)2 supplemented mitochondrial lysates showed presence of both dinitrosyl iron complexes and nitrosothiols. We suggest that nitric oxide attenuates production of mitochondrial superoxide by post-translational modifications by nitrosylation of protein cysteine residues and formation of protein dinitrosyl iron complexes with thiol-containing ligands and, therefore, nitric oxide reduction in pathological conditions associated with endothelial dysfunction may increase mitochondrial oxidative stress.

Left Ventricular Transmural Gradient in Mitochondrial Respiration Is Associated with Increased Sub-Endocardium Nitric Oxide and Reactive Oxygen Species Productions.

Objective: Left ventricle (LV) transmural gradient in mitochondrial respiration has been recently reported. However, to date, the physiological mechanisms involved in the lower endocardium mitochondrial respiration chain capacity still remain to be determined. Since, nitric oxide (NO) synthase expression in the heart has spatial heterogeneity and might impair mitochondrial function, we investigated a potential association between LV transmural NO and mitochondrial function gradient.Methods: Maximal oxidative capacity (VMax) and relative contributions of the respiratory chain complexes II, III, IV (VSucc) and IV (VTMPD), mitochondrial content (citrate synthase activity), coupling, NO (electron paramagnetic resonance), and reactive oxygen species (ROS) production (H2O2 and dihydroethidium (DHE) staining) were determined in rat sub-endocardium (Endo) and sub-epicardium (Epi). Further, the effect of a direct NO donor (MAHMA NONOate) on maximal mitochondrial respiratory rates (Vmax) was determined.Results: Mitochondrial respiratory chain activities were reduced in the Endo compared with the Epi (−16.92%; P = 0.04 for Vmax and –18.73%; P = 0.02, for Vsucc, respectively). NO production was two-fold higher in the Endo compared with the Epi (P = 0.002) and interestingly, increasing NO concentration reduced Vmax. Mitochondrial H2O2 and LV ROS productions were significantly increased in Endo compared to Epi, citrate synthase activity and mitochondrial coupling being similar in the two layers.Conclusions: LV mitochondrial respiration transmural gradient is likely related to NO and possibly ROS increased production in the sub-endocardium.

Salmonella enterica biofilm-mediated dispersal by nitric oxide donors in association with cellulose nanocrystal hydrogels.

Protected by extracellular polymers, microbes within biofilms are significantly more resistant to disinfectants. Current research has been instrumental in identifying nitric oxide donors and hydrogels as potential disinfectant additives. Nitric oxide (NO) donors are considered a very promising molecule as biofilm dispersal agents and hydrogels have recently attracted a lot of interest due to their biocompatible properties and ability to form stable thin films. When the NO donor MAHMA NONOate was dissolved in phosphate saline buffer, it was able to reduce the biomass of well-established biofilms up to 15% for at least 24 h of contact time. Encapsulation of MAHMA NONOate and molsidomine within a hydrogel composed of cellulose nanocrystals (CNC) has shown a synergistic effect in dispersing well-established biofilms: after 2 h of exposure, moderate but significant dispersion was measured. After 6 h of exposure, the number of cells transitioning from the biofilm to the planktonic state was up to 0.6 log higher when compared with non-treated biofilms. To further explore the transport processes of NO donors within hydrogels, we measured the nitric oxide flux from gels, at 25°C for a composite of 0.1 µM MAHMA NONOate–CNC. Nitric oxide diffuses up to 500 µm from the hydrogel surface, with flux decreasing according to Fick’s law. 60% of NO was released from the hydrogel composite during the first 23 min. These data suggest that the combined treatments with nitric oxide donor and hydrogels may allow for new sustainable cleaning strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-015-0114-7) contains supplementary material, which is available to authorized users.

Systematic analysis of the ability of Nitric Oxide donors to dislodge biofilms formed by Salmonella enterica and Escherichia coli O157:H7.

Biofilms in the industrial environment could be problematic. Encased in extracellular polymeric substances, pathogens within biofilms are significantly more resistant to chlorine and other disinfectants. Recent studies suggest that compounds capable of manipulating nitric oxide-mediated signaling in bacteria could induce dispersal of sessile bacteria and provide a foundation for novel approaches to controlling biofilms formed by some microorganisms. In this work, we compared the ability of five nitric oxide donors (molsidomine, MAHMA NONOate, diethylamine NONOate, diethylamine NONOate diethylammonium salt, spermine NONOate) to dislodge biofilms formed by non-typhoidal Salmonella enterica and pathogenic E. coli on plastic and stainless steel surfaces at different temperatures. All five nitric oxide donors induced significant (35-80%) dispersal of biofilms, however, the degree of dispersal and the optimal dispersal conditions varied. MAHMA NONOate and molsidomine were strong dispersants of the Salmonella biofilms formed on polystyrene. Importantly, molsidomine induced dispersal of up to 50% of the pre-formed Salmonella biofilm at 4°C, suggesting that it could be effective even under refrigerated conditions. Biofilms formed by E. coli O157:H7 were also significantly dispersed. Nitric oxide donor molecules were highly active within 6 hours of application. To better understand mode of action of these compounds, we identified Salmonella genomic region recA-hydN, deletion of which led to an insensitivity to the nitric oxide donors.

P32: Potential role for 8-Nitro-cGMP in nitrite-mediated hypoxic signaling

Background Nitrite has been shown to elicit both acute and chronic nitric oxide (NO)-dependent effects, especially at lower oxygen tensions.In this paradigm, nitrite reduction by deoxyhemoglobin and deoxymyoglobin activates NO-signaling to mediate several functions including hypoxic vasodilation.The mechanism underlying this however remains unclear, with a major challenge being how to reconcile nitrite-derived NO-signaling in an NO-scavenging environment, considering the high heme concentrations (∼10 mM) and rapid reaction rates between heme and NO ( k ∼ 10 7 M −1 s −1 ).Recent studies also implicate the derivative of cGMP, 8-nitro-cGMP, as a novel second messenger in NO-dependent signaling.Both cGMP and 8-nitro-cGMP activates PKG, however, unlike cGMP, 8-nitro-cGMP is resistant to PDE5 degradation and has the potential to elicit NO-induced signal transduction via a new chemical modification of proteins referred to as S -guanylation.To date, nitrite-dependent NO formation and 8-nitro-cGMP-dependent activation of NO-signaling have been considered independent of each other.In this study, we proposed a link between these two signaling paradigms.We hypothesized that 8-nitro-cGMP is a critical mediator of nitrite-dependent activation of hypoxic NO-signaling and tested if nitrite promotes hypoxic NO-signaling via 8-nitro-cGMP. Methods First, 8-nitro-cGMP formation in rat aorta exposed to nitrite at high O 2 (21%) and low O 2 (1%) tensions was assessed by immunostaining.The role of 8-nitro-cGMP in nitrite-mediated vasodilation was assessed using ex vivo aortic vessel dilation as an index of nitrite-dependent NO-signaling at high O 2 (21%) and low O 2 (1%) tensions, with and without the PDE5 inhibitors, sildenafil or zaprinast. Results The highest intensity of staining for 8-nitro-cGMP was observed in the vessels treated with nitrite at low O 2 (1%) tension and in the presence of RBC, indicating a nitrite-induced formation of 8-nitro-cGMP under hypoxic conditions.Nitrite stimulated vasodilation at 1% O 2 tension in a dose-dependent manner and this response was significantly improved in the presence of RBCs.Treatment of the aortic vessel segments with the PDE5 inhibitors, sildenafil or zaprinast improved RBC-dependent nitrite-mediated vasodilation by ∼2-fold at 21% O 2 .However, PDE5 inhibition had no effect on nitrite-dependent vasodilation at 1% O 2. Moreover, NO-dependent vasodilation using MAHMA NONOate was sensitive to PDE5 inhibition at both 21% and 1% O 2 .These data suggest a hypoxia-dependent shift from a PDE5-sensitive to PDE5-insensitive mechanism for nitrite-dependent vasodilation. Conclusion Collectively, these data indicate the possible role for 8-nitro-cGMP in nitrite-mediated hypoxic signaling paradigms. Disclosure Nothing to disclose.

Nitric oxide reduces Cl−absorption in the mouse cortical collecting duct through an ENaC-dependent mechanism

Since nitric oxide (NO) participates in the renal regulation of blood pressure, in part, by modulating transport of Na⁺ and Cl⁻ in the kidney, we asked whether NO regulates net Cl⁻ flux (JCl) in the cortical collecting duct (CCD) and determined the transporter(s) that mediate NO-sensitive Cl⁻ absorption. Cl⁻ absorption was measured in CCDs perfused in vitro that were taken from aldosterone-treated mice. Administration of an NO donor (10 μM MAHMA NONOate) reduced JCl and transepithelial voltage (VT) both in the presence or absence of angiotensin II. However, reducing endogenous NO production by inhibiting NO synthase (100 μM N(G)-nitro-L-arginine methyl ester) increased JCl only in the presence of angiotensin II, suggesting that angiotensin II stimulates NO synthase activity. To determine the transport process that mediates NO-sensitive changes in JCl, we examined the effect of NO on JCl following either genetic ablation or chemical inhibition of transporters in the CCD. Since the application of hydrochlorothiazide (100 μM) or bafilomycin (5 nM) to the perfusate or ablation of the gene encoding pendrin did not alter NO-sensitive JCl, NO modulates JCl independent of the Na⁺-dependent Cl⁻/HCO₃⁻ exchanger (NDCBE, Slc4a8), the A cell apical plasma membrane H⁺-ATPase and pendrin. In contrast, both total and NO-sensitive JCl and VT were abolished with application of an epithelial Na(+) channel (ENaC) inhibitor (3 μM benzamil) to the perfusate. We conclude that NO reduces Cl⁻ absorption in the CCD through a mechanism that is ENaC-dependent.

Optimal dosing regimen of nitric oxide donor compounds for the reduction of Pseudomonas aeruginosa biofilm and isolates from wastewater membranes

Membrane fouling by bacterial biofilms remains a key challenge for membrane-based water purification systems. Here, the optimal biofilm dispersal potential of three nitric oxide (NO) donor compounds, viz. sodium nitroprusside, 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine (MAHMA NONOate) and 1-(hydroxy-NNO-azoxy)-L-proline, disodium salt, was investigated using Pseudomonas aeruginosa PAO1 as a model organism. Dispersal was quantitatively assessed by confocal microscopy [bacterial cells and the components of the extracellular polymeric substances (EPS) (polysaccharides and extracellular DNA)] and colony-forming unit counts. The three NO donor compounds had different optimal exposure times and concentrations, with MAHMA NONOate being the optimal NO donor compound. Biofilm dispersal correlated with a reduction in both bacterial cells and EPS. MAHMA NONOate also reduced single species biofilms formed by bacteria isolated from industrial membrane bioreactor and reverse osmosis membranes, as well as in isolates combined to generate mixed species biofilms. The data present strong evidence for the application of these NO donor compounds for prevention of biofouling in an industrial setting.

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.

Intrinsic susceptibility of Giardia duodenalis assemblage subtypes AI, AII, B and EIII for nitric oxide under axenic culture conditions

The antigiardial effects of nitric oxide (NO·) have been reported in vitro, but only for assemblage A(I) lab strains. This study investigated the intrinsic NO· susceptibility of different assemblage subtypes. The susceptibility (IC₅₀) for NO· released by MAHMA NONOate was studied for three lab (WB, G1 and GS/M-83-H7) and six field isolates of assemblage subtypes A(I), A(II), B and E(III). Tests were performed in phosphate-buffered saline supplemented with L-cysteine HCl, trypticase peptone, powder bovine bile and 20% inactivated foetal calf serum (for assemblages A and E) or human serum (for assemblage B), adjusted to pH 7.3, to support adequate trophozoite survival. Flow cytometry with fluorescein diacetate and propidium iodide as viability indicators was used to determine trophozoite viability. This study indicated that the NO· susceptibilities of assemblage A lab and field strains (subtypes A(I) and A(II)) were fully comparable, indicating that the NO· susceptibility of the lab strains remained representative for their genotype. The trophozoites of assemblages B and E(III) showed comparable NO· susceptibilities that were markedly higher than the susceptibilities of assemblage subtypes A(I) and A(II). This study suggests a role for the assemblage subtype in defining NO· susceptibilities. The underlying mechanisms still need to be elucidated, but assemblage-linked differences in the expression of the genes coding for flavohemoglobin or A-type flavoprotein may certainly deserve further attention.

Impact of Systemically Active Neurohumoral Factors on the Erectile Response of the Rat

Mean arterial pressure (MAP) and specific regulation of penile blood flow are the primary determinants of an erection. While this concept is well recognized, the differential relationship between systemically acting vasoactive factors on arterial pressure and erectile responses is not well described. The aim of this study was to determine how the modification of systemic levels of neurohumoral factors impacts on the magnitude and efficiency of the erectile response. The main outcome measures for this study are changes in MAP and intracavernosal pressure (ICP) following electrostimulation of the cavernous nerve. Anesthetized adult, male Sprague-Dawley rats were catheterized for measuring MAP (carotid), ICP, and drug administration (vena cava). Erections were induced via cavernous nerve electrostimulation. Vasoactive drug infusions were used to produce changes in MAP levels including: hexamethonium, angiotensin II (ANGII)±hexamethonium, methoxamine±hexamethonium, losartan, MAHMA NONOate, and terbutaline. In general, ICP and MAP were linearly correlated regardless of treatment. Hexamethonium markedly dropped MAP and proportionately decreased the magnitude of the erectile response. ANGII or methoxamine given to hexamethonium-pretreated or untreated rats increased MAP similarly, but produced contrasting effects on erectile responses. ANGII-induced pressor responses were associated with increased erectile responses whereas all methoxamine treatments markedly decreased erectile responses. Depressor changes with losartan or terbutaline, but not MAHMA NONOate, also impacted negatively on the efficiency of the erectile responses at lower arterial pressures. In general, the magnitude of the erectile responses was found to be dependent upon the level of MAP, although the mechanism by which arterial pressure was changed impacted substantially on the characteristics of the relationship. The major finding was that circulation-wide α-adrenoceptor stimulation was extremely deleterious to erectile responses whereas global stimulation of ANG II receptors was actually proerectile. Overall, the results indicate that neurohumoral specificity in systemic hemodynamic control is also critical in establishing the optimal erectile environment in rats.

Long-lasting inhibition of presynaptic metabolism and neurotransmitter release by protein S-nitrosylation

Nitric oxide (NO) and related reactive nitrogen species (RNS) play a major role in the pathophysiology of stroke and other neurodegenerative diseases. One of the poorly understood consequences of stroke is a long-lasting inhibition of synaptic transmission. In this study, we tested the hypothesis that RNS can produce long-term inhibition of neurotransmitter release via S-nitrosylation of proteins in presynaptic nerve endings. We examined the effects of exogenous sources of RNS on the vesicular and nonvesicular L-[ 3H]glutamate release from rat brain synaptosomes. NO/RNS donors, such as spermine NONOate, MAHMA NONOate, S-nitroso-L-cysteine, and SIN-1, inhibited only the vesicular component of glutamate release with an order of potency that closely matched levels of protein S-nitrosylation. Inhibition of glutamate release persisted for > 1 h after RNS donor decomposition and washout and strongly correlated with decreases in the intrasynaptosomal ATP levels. Post-NO treatment of synaptosomes with thiol-reducing reagents decreased the total content of S-nitrosylated proteins but had little effect on glutamate release and ATP levels. In contrast, post-NO application of the end-product of glycolysis, pyruvate, partially rescued neurotransmitter release and ATP production. These data suggest that RNS suppress presynaptic metabolism and neurotransmitter release via poorly reversible modifications of glycolytic and mitochondrial enzymes, one of which was identified as glyceraldehyde-3-phosphate dehydrogenase. A similar mechanism may contribute to the long-term suppression of neuronal communication during nitrosative stress in vivo.

Inhibition of TRPC1/TRPC3 by PKG contributes to NO-mediated vasorelaxation

Nitric oxide (NO) inhibits transient receptor potential channel 3 (TRPC3) channels via a PKG-dependent mechanism. We sought to determine 1) whether NO inhibition of TRPC3 occurs in freshly isolated smooth muscle cells (SMC); and 2) whether NO inhibition of TRPC3 channels contributes to NO-mediated vasorelaxation. We tested these hypotheses in freshly isolated rat carotid artery (CA) SMC using patch clamp and in intact CA by vessel myograph. We demonstrated TRPC3 expression in whole CA (mRNA and protein) that was localized to the smooth muscle layers. TRPC1 protein was also expressed and coimmunoprecipitated with TRPC3. Whole cell patch clamp demonstrated nonselective cation channel currents that were activated by UTP (60 microM) and completely inhibited by a TRPC channel inhibitor, La(3+) (100 microM). The UTP-stimulated current (I(UTP)) was also inhibited by intracellular application of anti-TRPC3 or anti-TRPC1 antibody, but not by anti-TRPC6 or anti-TRPC4 control antibodies. We next evaluated the NO signaling pathway on I(UTP). Exogenous NO [(Z)-1-{N-methyl-N-[6(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA NONOate)] or a cell-permeable cGMP analog (8-bromo-cGMP) significantly inhibited I(UTP). Preapplication of a PKG inhibitor (KT5823) reversed the inhibition of MAHMA NONOate or 8-bromo-cGMP, demonstrating the critical role of PKG in NO inhibition of TRPC1/TRPC3. Intact CA segments were contracted with UTP (100 microM) in the presence or absence of La(3+) (100 microM) and then evaluated for relaxation to an NO donor, sodium nitroprusside (1 nM to 1 microM). Relaxation to sodium nitroprusside was significantly reduced in the La(3+) treatment group. We conclude that freshly isolated SMC express TRPC1/TRPC3 channels and that these channels are inhibited by NO/cGMP/PKG. Furthermore, NO contributes to vasorelaxation by inhibition of La(3+)-sensitive channels consistent with TRPC1/TRPC3.

Inhibition of TRPC3 by PKG contributes to NO‐mediated vasorelaxation

Nitric oxide (NO) inhibits heterologously expressed TRPC3 channels via PKG‐dependent phosphorylation. We examined whether NO inhibition of TRPC3 occurs in freshly isolated smooth muscle cells (SMC) and, if so, whether TRPC3 channel inhibition contributes to NO‐mediated vasorelaxation. We used rat carotid artery (CA) as our model tissue.Whole cell patch clamp of CA SMC demonstrated a non‐selective cation current that was activated by extracellular uridine triphosphate (UTP; 60 µM) and completely inhibited by La3+ (100 µM). The UTP‐stimulated current (IUTP) was also inhibited by anti‐TRPC3 antibody in the pipette solution, demonstrating that TRPC3 is the channel of IUTP. Application of an NO‐donor (MAHMA NONOate) or 8Br‐cGMP caused inhibition of IUTP. Pre‐incubation with a PKG inhibitor (KT5823) prevented inhibition by 8Br‐cGMP, demonstrating the essential role for PKG. Next, intact CA segments (in an artery myograph) were contracted with UTP (100 µM) in the presence or absence of La3+ (100 µM) and then relaxed with SNP, an NO donor. Relaxation to SNP was significantly less in the presence of La3+.In summary, we demonstrate that rat CA SMC expresses functional TRPC3 channels and that these channels are inhibited by the NO/cGMP/PKG signaling pathway. Furthermore, our data suggests that NO inhibition of TRPC3 contributes to NO‐mediated vasorelaxation. Supported by: AHA 0665100Y and NIH HL088435 (SPM)

Inhibition of anti‐IgE mediated human mast cell activation by NO donors is dependent on their NO release kinetics

Although the mast cell is a source of nitric oxide (NO), the effect of NO on human mast cells has not been defined. This study investigated if exogenous NO could affect human mast cell activation. Effects of different NO donors on immunoglobulin E (IgE)-dependent activation of human-cultured mast cells (HCMC) derived from precursors in buffy coat were investigated by measuring histamine release. Intracellular NO in HCMC was monitored with confocal microscopy using the fluorescent NO indicator 4-amino-5-methylamino-2', 7'-difluorofluorescein. Diethylamine NONOate (DEA/NO) and MAHMA NONOate (NOC-9), both have rapid NO release rates, only inhibited anti-IgE-induced histamine release when added to HCMC at the time of activation. NO donors with slower NO release kinetics were ineffective even after 30 min incubation. Confocal microscopy revealed that the effectiveness of NO donors was dependent on the availability of adequate NO inside HCMC during activation. The inhibitory action of DEA/NO was diminished by the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl but potentiated by the anti-oxidant, N-acetylcysteine (NAC). Furthermore, co-incubation with NAC allowed previously ineffective NO donors to suppress HCMC activation and thus suggested that NAC could increase the availability of NO from NO donors. Our results demonstrated that NO was able to modulate human mast cell activation but only when enough NO was present at the time of cell activation. Our findings explain the controversy over the effectiveness of NO on mast cell degranulation and supports the possibility that NO donors could be beneficial for treating allergic inflammation.

Acute depletion of heme by succinylacetone alters vascular responses but does not induce hypertension

Heme plays a critical role in blood pressure regulation because it is required by a number of enzymes that synthesize vascular modulators, including nitric oxide (NO), carbon monoxide (CO), guanosine 3',5'-cyclic monophosphate (cGMP), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin. The goal of this study was to examine the vascular effects of a short-term depletion of heme achieved through administration of the heme-synthesis inhibitor succinylacetone (SA), an irreversible inhibitor of aminolevulinic acid dehydratase (ALAD). Rats were depleted of heme by using a 4-day treatment with SA. This treatment impacted hemoenzyme function, decreasing renal nitric oxide synthase (NOS) activity (as indicated by decreased in vitro NOS activity), and increasing kidney microsomal heme oxygenase (HO) activity by 27%. SA treatment also resulted in enhanced reduction in blood pressure after infusions of exogenous NO donor MAHMA NONOate (at high dose) and acetylcholine (at low doses). Nevertheless, this SA treatment was insufficient to produce an overt elevation of basal arterial pressure. This latter lack of effect may be the result of multiple compensatory mechanisms for the regulation of blood pressure.

Nitric Oxide Stress Induces Different Responses but Mediates Comparable Protein Thiol Protection inBacillus subtilisandStaphylococcus aureus

The nonpathogenic Bacillus subtilis and the pathogen Staphylococcus aureus are gram-positive model organisms that have to cope with the radical nitric oxide (NO) generated by nitrite reductases of denitrifying bacteria and by the inducible NO synthases of immune cells of the host, respectively. The response of both microorganisms to NO was analyzed by using a two-dimensional gel approach. Metabolic labeling of the proteins revealed major changes in the synthesis pattern of cytosolic proteins after the addition of the NO donor MAHMA NONOate. Whereas B. subtilis induced several oxidative stress-responsive regulons controlled by Fur, PerR, OhrR, and Spx, as well as the general stress response controlled by the alternative sigma factor SigB, the more resistant S. aureus showed an increased synthesis rate of proteins involved in anaerobic metabolism. These data were confirmed by nuclear magnetic resonance analyses indicating that NO causes a drastically higher increase in the formation of lactate and butanediol in S. aureus than in B. subtilis. Monitoring the intracellular protein thiol state, we observed no increase in reversible or irreversible protein thiol modifications after NO stress in either organism. Obviously, NO itself does not cause general protein thiol oxidations. In contrast, exposure of cells to NO prior to peroxide stress diminished the irreversible thiol oxidation caused by hydrogen peroxide.

Uncoupling of Oxidative Phosphorylation in Vascular Function in Aging.

Aging is a risk factor for the development of CVD and is characterized by a reduced vascular reactivity and increased free radical generation (ROS). The uncoupling of oxidative phosphorylation (OXPHOS) reduces ROS generation. We hypothesized that endothelium-dependent (EDD) and endothelium-independent (EIND) dilation is linked to mitochondrial uncoupling. Isometric tension experiments were performed in Fisher 344 (F344, 15–16 months) rat aortic rings. Acetylcholine (Ach, 10 μM, n=13 rings) caused 10% vasodilation (n= 19). The EIND dilation was tested with MAHMA NONO-ate (10μM) and rings responded with 46% (n= 13, P<0.05) relaxation. FCCP (1 μM) a chemical uncoupler of OXPHOS had no effects on neither EDD nor EIND (n=6, ns). Bay 41–2272 (10 μM), stimulator of soluble guanylate cyclase (sGC) improved relaxation to Ach, and arteries responded with 56% (n=6, P<0.05) relaxation; relaxation to MAHMA was at 71% (n=6, P<0.05). The expression of uncoupling protein UCP-2 in heart and aorta of F344 was increased by 30% (3 hearts, 3 aortas, n=2), respectively; the expression of UCP-3 was increased by 3-fold (n=3) in F344 hearts as compare to age matched controls. Taken together, a decline in vascular function is consistent with progressive oxidative stress. The oxidative modification of proteins could be responsible for a lack of activation of a negative feedback via the uncoupling of OXPHOS and reduction in ROS levels.

Synthesis and Properties of a pH‐Insensitive Fluorescent Nitric Oxide Cheletropic Trap (FNOCT)

AbstractThe synthesis and properties of the new fluorescent nitric oxide cheletropic trap (FNOCT) 14, designed for the trapping and quantification of nitric oxide (NO) production in chemical and biological systems, is described (Scheme 3). The dicarboxylic acid 14 and the corresponding bis[(acetyloxy)methyl] ester derivative 15 of the FNOCT contain a 2‐methoxy‐substituted phenanthrene group as fluorophoric unit. The fluorescence of the reduced NO adduct of this FNOCT (λexc 320 nm, λem 380 nm) is pH‐independent. Trapping experiments were carried out in aqueous buffer solution at pH 7.4 with nitric oxide being added as a bolus as well as being released from the NO donor compound MAHMA NONOate (= (1Z)‐1‐{methyl[6‐(methylammonio)hexyl]amino}diazen‐1‐ium‐1,2‐diolate), indicating a trapping efficiency of ca. 50%. In a biological application, nitric oxide was scavenged from a culture of lipopolysaccharide‐stimulated rat alveolar macrophages. Under the applied conditions, a production of 11.1 ± 1.5 nmol of NO per hour and per 105 cells was estimated.