4,5‐diaminofluoroscein imaging of nitric oxide synthesis in crayfish terminal ganglia

We have analyzed the synthesis of nitric oxide in the terminal abdominal ganglion of the crayfish using the fluorescent probe 4,5‐Diaminofluoroscein diacetate, DAF‐2 DA. Following DAF‐2 loading, ganglia showed cell‐specific patterns of fluorescence in which the occurrence of strongly fluorescent cell bodies was highest in specific anterior, central, and posterior regions. We found that preincubation with the nitric oxide synthase (NOS) inhibitor L ‐NAME prevented much of the initial development of DAF‐2 fluorescence, whereas the inactive isomer D ‐NAME had no effect. Washout of preincubated L ‐NAME caused increased cell‐specific fluorescence due to endogenous NOS activity. Application of the NOS substrate L ‐arginine also resulted in an increase of DAF‐2 fluorescence in a cell‐specific manner. We bath applied the NO donor SNAP to increase exogenous NO levels which resulted in DAF‐2 fluorescence increases in most cells. We therefore presume that the cell‐specific pattern of DAF‐2 fluorescence indicates the distribution of neurones actively synthesizing NO. The similarity between the DAF‐2 staining pattern and previously published studies of NOS activity are discussed. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 361–369, 2002     

Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells

Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]_mt). Stimulation of mitochondrial Ca²⁺ uptake by exposure to different cytoplasmic Ca²⁺ concentrations (1, 2, and 5 µM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]_mt was sensitive to the NOS antagonist L-N⁵-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca²⁺ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl--cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca²⁺ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis. nitric oxide; nitric oxide synthase; calcium; endothelium; mitochondria     

Increased nitric oxide-dependent nitrosylation of 4,5-diaminofluorescein by oxidants: implications for the measurement of intracellular nitric oxide

4,5 diaminofluorescein (DAF-2) is increasingly utilized as a fluorescent detector for nitric oxide (*NO) in cells and tissues. In oxygenated solutions, reactive nitrogen species derived from (*) NO autoxidation nitrosate DAF-2 to yield the highly fluorescent DAF-2 triazole. In the present study, we investigated the nitrosation of DAF-2 at a neutral pH by absorption and fluorescence spectroscopy using NONOates as chemical sources of (*) NO. We found that both chemically synthesized peroxynitrite and horseradish peroxidase in the presence of hydrogen peroxide (H(2)O(2)) oxidized DAF-2 to a relatively stable nonfluorescent intermediate (t(1/2) approximately 90 s). Oxidation of DAF-2 prior to the addition of the z.rad;NO donor DEA/NO resulted in an increase in fluorescence that was approximately 7-fold higher than treatment with DEA/NO alone. The increase in DAF-2 triazole formation upon oxidation of DAF-2 was confirmed by high performance liquid chromatography. Peroxynitrite generated in situ from the equimolar production of (*) NO and superoxide (O(2)(*-)) also increased the yields of DAF-2 triazole formation, which was completely inhibited when O(2)(*-) was in excess of (*) NO. We propose that DAF-2 is oxidized to a free radical intermediate that directly reacts with (*) NO, thereby bypassing the requirement for (*)NO autoxidation for the formation of DAF-2 triazole. Our findings indicate that DAF-2 fluorometric assays are quantitatively difficult to interpret in cells and in solution when oxidants and (*) NO are co-generated.     

A fluorescence-based method for measuring nitric oxide in extracts of skeletal muscle.

We describe here a fluorescence assay for nitric oxide synthase activity in skeletal muscle based on a new indicator, 4,5-diaminofluorescein (DAF-2). The rapid and irreversible binding of DAF-2 to oxidized NO allows real-time measurement of NO production. The method is safer and more convenient than the usual citrulline radioassay and can be used with crude muscle extracts. Rabbit fast tibialis anterior (TA) muscle had a nitric oxide synthase (NOS) activity of 44.3 +/- 3.5 pmol/min/mg muscle. Addition of NOS blocker N(G)-allyl-L-arginine reduced this activity by 43%. Slow soleus muscle displayed NOS activity of 7.3 +/- 2.5 pmol/min/mg muscle, 16% that of the TA muscle. Continuous stimulation of TA muscle at 10 Hz for 3 weeks reduced NOS activity by 47% to an intermediate value consistent with the associated conversion of the muscle phenotype from fast to slow.     

Characterization of basal nitric oxide production in living cells

Nitric oxide (NO) is an important modulator of immune, endocrine and neuronal functions; however, measuring physiological levels of NO in cell cultures is generally difficult because of the lack of suitable methodologies. We have selected three cell lines from different origins: the neuroblastoma-derived Neuro2A (N2A), the cholinergic SN56 and the non-neuronal COS-1. We first demonstrated the presence of NADPH-diaphoretic activity, a potential marker of the NO-synthesizing (NOS) enzyme. By immunocytochemistry, using specific antibodies for each NOS subtype, we observed that subtype I was present in all cell lines and that subtype II was present in COS-1 and N2A cell lines. The presence of these NOS subtypes was further verified by Western blot analysis. Control cells treated with DAF-2 DA exhibited significant fluorescent levels corresponding to basal NO production. The subcellular distribution of the synthesizing enzyme was consistent with the NO-fluorescence signal; whereas, fixation affected the subcellular pattern of NO fluorescence signal. Addition of NOS inhibitors or NO scavengers to the incubation medium reduced the intensity of the NO fluorescence signal in a concentration-dependent manner. Conversely, increasing concentrations of a NO donor, or incident light, increased the fluorescence intensity. Our observation of NO production and distribution using the DAF-2 method has a direct impact on studies using these cell lines.     

Modulation of nitric oxide (NO) biosynthesis in lactobacilli

We characterized effects of nitric oxide synthase (NOS) substrate L-arginine and classical inhibitors of mammalian NOS on nitric oxide (NO) biosynthesis in probiotic bacteria Lactobacillus plantarum 8P-A3. NO-synthase origin of nitric oxide detected by fluorescent NO indicator 1,2-diaminoanthraquinone (DAA) was confirmed by induction of NO production by exogenous L-arginine. None of the used inhibitors of three isoforms of mammalian NOSs (L-NAME, L-NIL, nNOS inhibitor I) showed significant inhibitory effect of lactobacillar NO-synthase activity.     

Simultaneous measurement of intracellular Ca(2+) and nitric oxide: a new method

Different methods to measure the unstable radical nitric oxide (NO) have been established. We are going to present a new method to measure intracellular calcium and NO simultaneously in endothelial cells. A new fluorescent dye (DAF-2) has been developed recently which binds NO resulting in an enhanced fluorescence. We loaded porcine aortic endothelial cells with Fura-2, a fluorescent dye commonly used to measure intracellular calcium, and DAF-2 simultaneously (cell permeable dyes). Using excitation wavelengths of lambda 340 nm (Fura-2) and lambda 485 nm (DAF-2) we could show that thrombin induces an intracellular calcium increase and simultaneously a NO formation in endothelial cells which could be blocked by a NO synthase inhibitor. This new method of a simultaneous measurement of intracellular calcium and NO provides the possibility to follow intracellular calcium and NO distributions online, and is sensitive enough to monitor changes of NO formed by the constitutive endothelial NO-synthase.     

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).     

Nitric oxide synthase in filariae: demonstration of nitric oxide production by embryos in Brugia malayi and Acanthocheilonema viteae

The radical gas nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from l-arginine and molecular oxygen. Nitric oxide is an important signaling molecule in invertebrate and vertebrate systems. Previously we have shown that NOS is localized to more tissues in Brugia malayi than has been reported in Ascaris suum. In this paper, we analyze the distribution of NOS in Acanthocheilonema viteae, a filarial nematode that differs from B. malayi in that A. viteae females release microfilariae without a sheath. A. viteae is also one of a few filarial parasites without the Wolbachia intracellular endosymbiont. By use of a specific antibody, NOS was demonstrated in extracts of A. viteae and Dirofilaria immitis. The localization pattern of NOS in A. viteae was similar to that seen in B. malayi, with the enzyme localized to the body wall muscles of both sexes, developing spermatozoa, intrauterine sperm, and early embryos. By use of DAF-2, a fluorescent indicator specific for nitric oxide, the embryos of B. malayi and A. viteae were demonstrated to produce NO ex utero. The near identical staining patterns seen in A. viteae and B. malayi argue that NO is not produced by Wolbachia, nor is it produced by the nematodes in response to the infection. Localization of NOS to the sperm of filarial nematodes suggests a role for NO during fertilization as has been described for sea urchin and ascidian fertilization. Demonstration of the activity of embryonic NOS supports our earlier hypothesis that NO is a signaling molecule during embryogenesis in filarial nematodes.     

Determinants of shear stress-stimulated endothelial nitric oxide production assessed in real-time by 4,5-diaminofluorescein fluorescence

The extremely short biological half-life of endothelial-derived nitric oxide (NO) has impeded real-time measurements of NO synthesis. We used the membrane-permeable fluorescent probe 4,5-diaminofluorescein diacetate (DAF-2 DA) to study determinants of NO synthesis in bovine aortic endothelial cells (BAECs). A step increase in shear stress (SS) from 0.3 to 3.4 dyne/cm(2) triggered an increase in DAF-2 fluorescence starting 3.0 +/- 0.5 min after the flow rise and peaking at 44.7 +/- 7.2 min. This was abolished by intracellular Ca(2+) chelation, but was unaffected by blocking extracellular Ca(2+) influx or by inhibiting SS-related changes in intracellular pH. The increase in DAF-2 fluorescence occurred significantly earlier in BAECs transfected with either superoxide dismutase (SOD) or catalase (CAT), indicating concomitant reactive oxygen species (ROS) generation by SS and "competition" between ROS- and DAF-2-NO interactions. These data provide novel insights into several NO signaling determinants and reveal that DAF-2 can assess real-time SS-stimulated NO synthesis in endothelial cells. This should facilitate the analysis of NO-signaling pathways.     

Pitfalls and limitations in using 4,5-diaminofluorescein for evaluating the influence of polyphenols on nitric oxide release from endothelial cells

The reagent 4,5-diaminofluorescein (DAF-2) is a widely utilized and sensitive fluorescent probe for real-time assessment of nitric oxide (NO) production. In this study we investigated the feasibility of using DAF-2 for detection of NO release from EA.hy 926 human endothelial cells stimulated with plant polyphenols. Flavonoids have recently gained much interest because of reported beneficial effects on vasodilatation, which have been ascribed to stimulation of endothelial NO production. DAF-2 shows moderate fluorescence, and because certain phenolic compounds quench fluorescence or fluoresce themselves, we utilized liquid chromatography to avoid interference. Our investigations with (+)-catechin and trans-resveratrol as test phenolic compounds revealed various previously undescribed principal methodologic pitfalls and limitations. Under assay conditions (+)-catechin displayed a highly significant increase in fluorescence intensity so that a control of test compound stability is advisable. Moreover, DAF-2 was subject to conversion to triazolofluorescein (DAF-2T) under certain assay and storage conditions; thus control of spontaneous reagent conversion is advisable. Finally, formation of DAF-2T was dose-dependently inhibited by polyphenols to a degree consistent with their free radical scavenging activity. The inhibition of DAF-2T generation seems to contradict previous reports on enhanced NO release from endothelial cells by (+)-catechin and resveratrol. Therefore, the planning of experiments involving NO measurement in biological systems and interpretation of results requires substantial scrutiny.     

Physical conditioning modulates rat cardiac vascular endothelial growth factor gene expression in nitric oxide-deficient hypertension

Many individuals with cardiac diseases undergo periodic physical conditioning with or without medication to improve cardiovascular health. Therefore, this study investigated the interaction of physical training and chronic nitric oxide synthase (NOS) inhibitor (nitro-L-arginine methyl ester, L-NAME) treatment on blood pressure (BP), cardiac vascular endothelial factor (VEGF) gene expression, and nitric oxide (NO) systems in rats. Fisher 344 rats were divided into four groups and treated as follows: (1) sedentary control, (2) exercise training (ET) for 8 weeks, (3) L-NAME (10mg/kg, s.c. for 8 weeks), and (4) ET+L-NAME. BP was monitored with tail-cuff method. The animals were sacrificed 24h after last treatments and hearts were isolated and analyzed. Physical conditioning significantly increased respiratory exchange ratio, cardiac NO levels, NOS activity, endothelial eNOS, and inducible iNOS protein expression as well as VEGF gene expression. Training also caused depletion of cardiac malondialdehyde (MDA) levels indicating the beneficial effects of the training. Chronic L-NAME administration resulted in a depletion of cardiac NO level, NOS activity, and eNOS, nNOS, and iNOS protein expressions, as well as VEGF gene expression (2-fold increase in VEGF mRNA). Chronic L-NAME administration also enhanced cardiac MDA levels indicating cardiac oxidative injury. These biochemical changes were accompanied by increases in BP after L-NAME administration. Interaction of training and NOS inhibitor treatment resulted in normalization of BP and up-regulation of cardiac VEGF gene expression. The data suggest that physical conditioning attenuated the oxidative injury caused by chronic NOS inhibition by up-regulating the cardiac VEGF and NO levels and lowering the BP in rats.     

Opposing effects of coupled and uncoupled NOS activity on the Na+-K+ pump in cardiac myocytes

Pharmacological delivery of nitric oxide (NO) stimulates the cardiac Na(+)-K(+) pump. However, effects of NO synthesized by NO synthase (NOS) often differ from the effects of NO delivered pharmacologically. In addition, NOS can become "uncoupled" and preferentially synthesize O(2)(.-), which often has opposing effects to NO. We tested the hypothesis that NOS-synthesized NO stimulates Na(+)-K(+) pump activity, and uncoupling of NOS inhibits it. To image NO, we loaded isolated rabbit cardiac myocytes with 4,5-diaminofluorescein-2 diacetate (DAF-2 DA) and measured fluorescence with confocal microscopy. L-arginine (L-arg; 500 micromol/l) increased DAF-2 DA fluorescence by 51% compared with control (n = 8; P < 0.05). We used the whole cell patch-clamp technique to measure electrogenic Na(+)-K(+) pump current (I(p)). Mean I(p) of 0.35 +/- 0.03 pA/pF (n = 44) was increased to 0.48 +/- 0.03 pA/pF (n = 7, P < 0.05) by 10 micromol/l L-Arg in pipette solutions. This increase was abolished by NOS inhibition with radicicol or by NO-activated guanylyl cyclase inhibition with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. We next examined the effect of uncoupling NOS using paraquat. Paraquat (1 mmol/l) induced a 51% increase in the fluorescence intensity of O(2)(.-)-sensitive dye dihydroethidium compared with control (n = 9; P < 0.05). To examine the functional effects of uncoupling, we measured I(p) with 100 micromol/l paraquat included in patch pipette solutions. This decreased I(p) to 0.28 +/- 0.03 pA/pF (n = 12; P < 0.001). The paraquat-induced pump inhibition was abolished by superoxide dismutase (in pipette solutions). We conclude that NOS-mediated NO synthesis stimulates the Na(+)-K(+) pump, whereas uncoupling of NOS causes O(2)(.-)-mediated pump inhibition.     

Inhibitors of nitric oxide synthase attenuate nerve growth factor-mediated increases in choline acetyltransferase expression in PC12 cells

NGF can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. To investigate the role of NO in NGF-activated expression of cholinergic phenotype, PC12 cells were treated with either the nonselective NOS inhibitor L-NAME (N (omega)-nitro-L-arginine methylester) or the inducible NOS selective inhibitor MIU (s-methylisothiourea), and the effect on NGF-stimulated ChAT mRNA levels and ChAT specific activity was determined. NGF increased steady-state levels of mRNA and protein for both inducible and constitutive isozymes of NOS in PC12 cells, and led to enhanced NOS activity and NO production. MIU and, to a lesser extent, L-NAME blocked neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. Both L-NAME and MIU attenuated NGF-mediated increases in choline transferase (ChAT)-specific activity and prevented the increase in expression of ChAT mRNA normally produced by NGF treatment of PC12 cells. The present study indicates that NO may be involved in the modulation of signal transduction pathways by which NGF leads to increased ChAT gene expression in PC12 cells.     

Exercise conditioning attenuates the hypertensive effects of nitric oxide synthase inhibitor in rat

Many individuals with cardiovascular diseases undergo periodic exercise conditioning with or with out medication. Therefore, this study investigated the interaction of exercise training and chronic nitric oxide synthase (NOS) inhibitor (Nitro-L-Arginine Methyl Ester, L-NAME) treatment on blood pressure and its correlation with aortic nitric oxide (NO), antioxidant defense system and oxidative stress parameters in rats. Fisher 344 rats were divided into four groups: (1) sedentary control, (2) exercise training (ET) for 8 weeks, (3) L-NAME (10 mg/kg, subcutaneous for 8 weeks) and (4) ET + L-NAME. Blood pressure (BP) was monitored weekly for 8 weeks with tail-cuff method. The animals were sacrificed 24 h after last treatments and thoracic aortic rings were isolated and analyzed. Exercise conditioning resulted in a significant increase in respiratory exchange ratio (RER), aortic NO production, NO synthase activity and inducible iNOS protein expression. Training significantly enhanced aortic GSH levels, GSH/GSSG ratio and up-regulation of aortic CuZn-SOD, Mn-SOD, catalase (CAT) glutathione peroxidase (GSH-Px) activity and protein expression and significantly decreased aortic lipid peroxidation. Chronic L-NAME administration resulted in a significant depletion of aortic NO, NOS activity, endothelial (eNOS) and iNOS protein expression, GSH level, GSH/GSSG ratio, down-regulation of aortic antioxidant enzyme activities and protein expressions. Aortic xanthine oxidase (XO) activity significantly increased with increased lipid peroxidation and protein oxidation after L-NAME administration. The biochemical changes were accompanied by increased in BP. Interaction of training and chronic NOS inhibitor treatment resulted in normalization of BP and aortic antioxidant enzyme activity and protein expression, up-regulation of aortic GSH/GSSG ratio, NO levels, Mn-SOD protein expression, depletion of GSSG, protein oxidation and lipid peroxidation. The data suggest that training attenuated the oxidative injury caused by chronic NOS inhibitor treatment by up-regulating the NO and antioxidant systems and lowering the BP in rats.     

Reliable in vitro measurement of nitric oxide released from endothelial cells using low concentrations of the fluorescent probe 4,5-diaminofluorescein

4,5-Diaminofluorescein (DAF-2) and its membrane-permeable derivate DAF-2 diacetate are fluorescent probes that have been developed to perform real-time biological detection of nitric oxide (NO). Their use for intracellular imaging, however, has recently been seriously questioned and data using DAF-2 for extracellular NO detection at low levels, as for example released from endothelial cells, are rare. Here we show that a reliable detection of low levels of NO in biological systems by DAF-2 is possible (a) by using low DAF-2 concentrations (0.1 microM) and (b) by subtracting the DAF-2 auto-fluorescence from the measured total fluorescence. The described method allows easy real-time detection of endothelial NO formation.     

Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2): implications for cellular NO imaging

The fluorescent indicator of nitric oxide (NO), 4,5-diaminofluorescein (DAF-2), and its membrane-permeable derivative (DAF-2 diacetate) have been recently developed to perform real-time biological imaging of NO. In this study, we show that DAF-2 is strongly influenced by factors other than the concentration of NO itself. Using measurements with a fluorimeter as well as fluorescence microscopy, we found that the divalent cation concentration in the medium, as well as the incident light, strongly affects the ability of DAF-2 to detect NO. Calcium, in particular, enhanced the signal detection of NO released by NO donors by up to 200 times. With multiple and longer exposures to light, no bleaching of the dye was observed but, instead, a potentiation of the fluorescence response could be measured. While these two properties will affect the use and interpretation of the hitherto acquired data with this fluorescent compound, they may also open up new possibilities for its application.     

Nitric oxide spatial distribution in single cultured hippocampus neurons: investigation by projection of reconstructed 3-D image and visualization technique

Recent studies have revealed a non-homogeneous distribution of nitric oxide synthase (NOS) in neurons. However, it is not yet clear whether the intracellular distribution of NOS represents the intracellular nitric oxide (NO) distribution. In the present study, software developed in our laboratory was applied to the reconstructed image obtained from confocal slice images in order to project the 3-D reconstructed images in any direction and to cut the neuron in different sections. This enabled the spatial distribution of NO to be visualized in any direction and section. In single neurons, NO distribution was seen to be heterogeneous. After stimulation with glutamate, the spatial changes in different areas of the neuron were different. These findings are consistent with immunocytochemical data on the intracellular localization of nNOS in hippocampus neurons, and will help to elucidate the specificity of nitric oxide signaling. Finally, the administration of SNAP and L-NAME was used to examine DAF-2 distribution in the neurons. The results showed this distribution to be homogenous; therefore, it did not account for the NO distribution results.