Activity of deoxyribonucleoside-activated nucleotidase in cells from various lymphoid mouse tissues and in concanavalin A-stimulated lymphocytes

The specific activity in cells from lymph nodes, spleen and thymus was 32, 28 and 25 nmol/min per mg of cytosol protein, respectively, whereas that in bone marrow cells was significantly lower (10 units/mg of protein). No difference in specific DAN activity between isolated B- and T-lymphocytes was observed. Two types of lymphoid mouse cell lines (MOPC-31C plasmacytoma cells, S49 Cyc- lymphoma cells) showed specific activities similar to the normal lymphoid cells. In concanavalin A- stimulated spleen lymphocytes in culture there was a rapid increase in DAN activity shortly after maximum DNA synthesis, reaching a plateau 2-3 times the original level. The enzyme (DAN) of mouse tissues possessed the characteristic properties previously detected for the rat enzyme.     

Biochemical properties and immunohistochemical localization of carbonic anhydrase in the sacculus of the inner ear in the salmon Oncorhynchus masou

Carbonic anhydrase (CA) in the inner ear sacculus was examined by activity assay, Western blotting and immunohistochemistry to determine its role in otolith calcification. An immunoreactive protein with a molecular mass of approximately 28 kDa was detected by Western blotting. The CO2 hydration activity in the cytosol fraction of the sacculus was 5.4 units/mg protein, while little or no activity was detected in the nuclear and mitochondrial fractions. The enzyme activity was highly inhibited by acetazolamide. The concentration of 50% inhibition was 8.16 nM and the inhibition constant of the activity was 8.25 nM. Transitional and squamous epithelial cells of the sacculus were immunopositive with an anti-CA II antibody, but sensory epithelial cells and mitochondria-rich cells in the transitional epithelium were not. These results suggest that transitional epithelial cells other than mitochondria-rich cells and squamous epithelial cells play an important role in otolith calcification by supplying bicarbonate to otoliths and/or by eliminating protons from endolymph.     

Measurement and activity of cytosolic deoxyribonucleoside-activated nucleotidase in various cell types from rat liver and spleen

The enzyme activity was measured in hepatocytes, Kupffer cells, endothelial cells and spleen cells. Hepatocytes showed proportionality between enzyme activity and cytosol concentration, but with Kupffer cells, endothelial cells and spleen cells the specific activity decreased with decreasing cytosol concentration when the amount of cytosol protein in 250 microliters incubation mixture was below 80, 60 and 20 micrograms, respectively. The specific activities in hepatocytes, Kupffer cells, endothelial cells and spleen cells were 2, 16, 18 and 115 nmol/min per mg of cytosol protein, respectively.     

Partial characterization and ontogeny of renal cytosolic glucocorticoid receptors in mouse kidney

The glucocorticoid receptor (GR) was partially characterized in mouse renal cytosol. A sensitive and reproducible [3H]dexamethasone binding assay suitable for use with small quantities of cytosolic protein, was developed. Studies defined the optimal equilibrium binding conditions, metabolism of [3H]dexamethasone in adult renal cytosol, specificity of binding of the GR, and molecular weight of the GR-[3H]dexamethasone complex by gel filtration chromatography. The assay was subsequently used to measure the renal GR during different stages of foetal and postnatal development, as well as in glomerular and renal tubular preparations from adult mice. An almost linear increase in GR occurred from day 13 to day 18 of gestation with levels rising from 100 to 201 fmol/mg cytosol protein; this was followed by a sharp rise in receptor concentration just after birth to 343 fmol/mg cytosol protein. Adult levels, 410-433 fmol/mg cytosol protein, were reached by 2 weeks after birth. The equilibrium dissociation constants (Kd) of the [3H]dexamethasone-receptor complex were similar in adult and in embryonic cytosols (range, 2.8-11.8 nM; mean +/- SD = 6.5 +/- 2.9 nM). Specific binding was assessed to be 3- to 5-fold greater in tubular than in glomerular preparations. These data on the localization and ontogeny of GR during murine metanephric development provide a basis for study of glucocorticoid-mediated effects on various models of congenital and acquired renal disease.     

Nitrate and nitrite reduction in the plant fraction of alfalfa root nodules

The plant fraction of alfalfa ( Medicago sativa L. cv. Aragon) nodules contained both nitrate reductase (NR) and nitrite reductase (NiR). Specific activity of NADH-NR from the cytosol of nodules not treated with NO₃- was about 30 nmol (mg protein)^-1-h^-1 and was not basically affected by NO₃ addition. In contrast, typical specific activity for cytosolic NiR was 1.5 umol (mg protein)^-1h^-1 using methyl viologen as electron donor. This activity strongly increased with NO₃ concentration, probably due to substrate induction. Maximal activity was 3.5 μmol (mg protein)^-1h^-1 at 50 to 200 mM NO₃.
Estimates indicate that the contribution of cytosol to the overall NR and NiR activities of alfalfa nodules is distinctly different: less than 10% and about 70%, respectively. The increasing amounts of NO₂ accumulating in the cytosol upon NO₃, supply, and the different response to NO₃ of bacteroid and cytosolic NRs support the concept that most of this NO₂ comes from the bacteroids.     

Interaction of membranes of the Golgi complex with microtubules in vitro.

We have developed an in vitro assay for characterizing the binding of elements of the Golgi complex to microtubules. The binding assay comprises three distinct components, Golgi elements purified from Vero cells by subcellular fractionation, taxol-polymerized tubulin from bovine brain coupled to magnetic beads and cytosol from HeLa cells. Binding of Golgi elements to microtubules is quantitated by measuring the activity of the Golgi marker enzyme, galactosyltransferase, associated with the microtubule-coated beads retrieved with a magnet. In the presence of cytosol, 35 to 45% of the total input of galactosyltransferase activity (Golgi elements) bind to microtubules; only 3% of the Golgi elements bind to microtubules, however, in the absence of cytosolic factors. This binding is saturable at a cytosol concentration of approximately 5 mg/ml or at a high input of Golgi elements. Cytosol-stimulated binding of Golgi elements to microtubules is decreased to less than 15% when cytosol is pretreated with 2 mM N-ethylmaleimide (NEM) and it is abolished when cytosolic proteins are inactivated by heat or when microtubules have been coated with heat-stable microtubule-associated proteins (MAPs). Trypsinization of the membranes of the Golgi elements abolishes their ability to bind to microtubules. Furthermore, inactivation of cytoplasmic dynein by UV/vanadate treatment does not affect the binding. This suggests that the interaction of Golgi elements with microtubules depends on NEM-sensitive cytosolic factors and membrane-associated receptors, but not on the microtubule-based motor protein cytoplasmic dynein.     

Calmodulin activity in whole body and fat body tissue extracts of Heliothis virescens larvae

Calmodulin is an activator of many enzymatic activities. Total calmodulin activity in tissue extracts of Heliothis virescens larvae (5th instar), assayed by cyclic phosphodiesterase activation, was 0.48 unit/gm for whole body and 22.2 units/gm for fat body. Specific calmodulin activity was 0.1 unit/mg protein for whole body and 3.0 units/mg protein for fat body. The larval fat body is therefore the main site of calmodulin activity in this lepidopterous larva.     

Aromatase activity in microsomal preparations of human genital skin fibroblasts: influence of glucocorticoids

Skin is an important site of estrogen production in men. Although the aromatase complex in these cells appears to be similar to that of other human cells, the regulation of aromatase by glucocorticoids in cultured human skin fibroblasts is unique. We examined aromatase activity in microsomal-enriched fractions of cultured human skin fibroblasts in order to characterize better the factors that regulate the aromatase in these cells. The optimum pH for aromatase activity in microsomal preparations ranged between 7.0 and 7.5. When androstenedione was the substrate, the mean Vmax was 0.58 pmol/mg protein/h (range: 0.09-1.26 pmol/mg protein/h) and the mean Km was 27 nM (range: 9-50 nM). When aromatase activity was determined as a function of NADPH concentration, the mean Vmax was 0.39 pmol/mg protein/h (range 0.11-0.82 pmol/mg protein/h) and the mean Km was 180 microM (range: 86-300 microM). For skin fibroblasts exposed to DEX, aromatase activity in isolated microsomes and intact cells was stimulated demonstrating a typical time course with peak levels at 14h and a decline toward baseline with prolonged (48-60 h) exposure. Cytosol from DEX-stimulated cells did not stimulate the aromatase activity in microsomal-enriched preparations from untreated cells. In addition, cytosol from cells incubated with DEX for a prolonged period (60 h) did not inhibit the higher aromatase activity of microsomes from cells incubated with DEX for only 14 h. We previously demonstrated that skin fibroblasts incubated with DEX and CHX produced a superinduction phenomenon for aromatase activity. This superinduction of enzyme activity also occurred in the microsomal-enriched fraction and was unaffected by the cytosol of these cells. These studies exclude the possibility that the unique effects of DEX on the aromatase in human skin fibroblasts are due to the production of either inhibitory or stimulatory soluble factors within cytosol.     

Subcellular distribution and activities of superoxide dismutase,catalase, glutathione peroxidase,and glutathione reductase in the southern armyworm,Spodoptera eridania

In mid-fifth-instar larvae of the southern armyworm, Spodoptera eridania, the subcellular distribution of four antioxidant enzymes—superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX), and glutathione reductase (GR)—were examined. Two-thirds (4.26 units ·mg protein^?1) of the SOD activity was found in the cytosol, and one-thirds (2.13 units ·mg protein^?1) in the mitochondria. CAT activity was unusually high and not restricted to the microsomal fraction where peroxisomes are usually isolated. The activity was distributed as follows: cytosol (163 units) mitochondria (125 units) and microsomes (119 units). Similar to CAT, the subcellular compartmentalization of both GPOX and GR was unusual. No activity was detected in the cytosol, but in mitochondria and microsomes, GR levels were 5.49 and 3.09 units. Although GPOX activity exhibited 14–16-fold enrichment in mitochondria and microsomes, respectively, over the 850g crude homogenate, the level was negligible (mitochondria = 1.4 × 10^?3 units; microsomes = 1.6 × 10^?3 units), indicating that this enzyme is absent. The unusual distribution of CAT has apparently evolved as an evolutionary answer to the absence of GR from the cytosol, and the lack of GPOX activity.     

p73-dependent expression of DAN during cisplatin-induced cell death and osteoblast differentiation

We previously reported that DAN, a founding member of the DAN family of secreted proteins, acts as an inhibitor of cell cycle progression and is closely involved in retinoic acid-induced neuroblastoma differentiation. In this study, we found that DAN as well as p73, the recently identified p53 family member, was up-regulated during osteoblast differentiation. Additionally, the expression of DAN was increased in response to cisplatin-induced cell death of neuroblastoma SH-SY5Y cells. Consistent with the previous reports, p73 was accumulated after the treatment with cisplatin. Intriguingly, we found a putative p53/p73-binding site in the 5'-upstream region of the human DAN gene. A luciferase reporter assay and an in vitro DNA-binding experiment revealed that this canonical p53/p73-binding site was a functional responsive element and was specific for p73. Our results suggest that there exists a functional association between DAN and p73 during osteoblast differentiation as well as cisplatin-induced cell death.     

Synthesis of nitrogen oxides from L-arginine by macrophage cytosol: requirement for inducible and constitutive components

Cytosols prepared from murine peritoneal macrophages and the RAW 264 macrophage cell line catalyzed conversion of L-arginine to the labile vaso-relaxant nitric oxide and its accumulating endproducts, nitrite and nitrate. This activity required previous exposure of the cells to interferon-gamma and bacterial lipopolysaccharide. Nitrogen oxide synthetase activity was characterized further using nitrite + nitrate production as an indicator of the synthesis of all three nitrogen oxides. Nitrogen oxide synthetase activity was heat-sensitive, NADPH-dependent, and exhibited substrate stereospecificity. The nitrite + nitrate formation was proportional to time and concentration of cytosol. However, dilution decreased the specific activity, suggesting a cofactor requirement in addition to NADPH. Specific activity was restored by addition of cytosol from non-activated macrophages, which itself did not make nitric oxide. Both high and low molecular weight fractions of control macrophage cytosol were required to restore activity of cytosol from activated macrophages that had been either diluted or partially purified. Thus, the enzymatic system involved in nitric oxide synthesis by murine macrophages consists of at least one inducible and two constitutive components.     

Steroid metabolism and binding activity in a murine renal tumor cell line

The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextran-coated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific 5 alpha-dihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, R1881 and ORG2058 all completely inhibited [3H]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-filter assay, with an estimated Kd for methyltrienolone (R1881) of 5 nM at 0 degrees C. Scatchard analysis of [3H]Dx binding by RAG cytosol showed a Kd of 6 nM for Dx and 44 nM for corticosterone at 0 degrees C. Glucocorticoid receptor levels were estimated to be 182 fmol/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [3H]testosterone and [3H]DHT by RAG cells was examined 1, 4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [3H]testosterone. There was little conversion of DHT to androstanediol.     

Coupled-enzyme and direct assays for uroporphyrinogen III synthase activity in human erythrocytes and cultured lymphoblasts: Enzymatic diagnosis of heterozygotes and homozygotes with congenital erythropoietic porphyria

Rapid and reproducible assays for uroporphyrinogen III synthase (URO-S; EC 4.2.1.75) have been developed and used to determine the enzymatic activity in human erythrocytes and cultured lymphoid cells. In the coupled-enzyme assay, porphobilinogen was first converted to hydroxymethylbilane, the natural substrate for URO-S, by hydroxymethylbilane synthase which was conveniently obtained from heat-treated erythrocyte lysates. In the direct assay, synthetic hydroxymethylbilane was used as substrate. In both assays, the uroporphyrinogen reaction products were oxidized to their respective uroporphyrin isomers, which were then resolved and quantitated by reversed-phase high-pressure liquid chromatography. Both assays were optimized for pH, substrate concentration, and linearity with time and protein concentration. The mean URO-S activities in normal human erythrocyte lysates determined by the coupled-enzyme and direct assays were 7.41 ± 1.35 and 7.64 ± 1.73 units/mg protein, respectively. In normal human cultured lymphoid cells, the mean activities were 13.7 ± 1.39 and 17.6 ± 1.15 units/mg protein for the coupled-enzyme and direct assays, respectively. In four families with congenital erythropoietic porphyria, both assays reliably identified the markedly decreased URO-S activities in erythrocytes and cultured lymphoid cells from affected homozygotes and the half-normal activities in these sources from obligate heterozygotes. The coupled-enzyme assay was easier to perform and was suited for clinical diagnostic assays and for monitoring enzyme purification procedures, while the direct assay, which required substrate preparation and technical dexterity, was best for kinetic studies of URO-S.     

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

Chromatin-associated ornithine decarboxylase at early stages of growth and differentiation of etiolated mono- and dicotyledonous plants

Ornithine decarboxylase (ODC) of barley, corn, bean and pea plants was associated with chromatin at early stages of growth. In corn, bean and pea plants the chromatin of roots possessed ODC with the highest specific activity of 30–65 units per mg protein. After 200 h of growth ODC activity of chromatin declined, while ODC activity of cytosol increased linearly to 4–19 units per mg protein. ODC activity of either chromatin or cytosol of shoots in the above mentioned plants was usually low, 1–2 units per mg protein and only at the beginning of shoot growth (96 h) did pea chromatin have high ODC activity (14 units per mg protein). In these plants ODC was tightly bound to chromatin and could not be extracted with different concentrations of NaCl (0.1–1.0 M) or non-ionic detergent (Triton X100, Tween 20) but could be extracted by freezing and thawing with satisfactory recovery.     

Mutation and analysis of Dan, the founding member of the Dan family of transforming growth factor beta antagonists

The Dan family of transforming growth factor beta antagonists is a large, evolutionarily conserved family of proteins. Little is known about either the specificity of these antagonists or the biological roles of these proteins. We have characterized Dan, the founding member of this family, with regard to both its biochemical specificity and its biological roles. Although DAN is not an efficient antagonist of BMP-2/4 class signals, we found that DAN was able to interact with GDF-5 in a frog embryo assay, suggesting that DAN may regulate signaling by the GDF-5/6/7 class of BMPs in vivo. Intriguingly, in developing neurons, Dan mRNA was localized to axons, suggesting a potential role for the DAN protein in axonal outgrowth or guidance. Mice lacking Dan activity were generated by gene targeting and displayed subtle, background-dependent defects.     

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by incubation of rat hepatocytes with phospholipase A2

The effect of phospholipase A2 treatment of rat hepatocytes on CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis was investigated. Cytidylyltransferase is recovered from the cytosol and in a membrane-bound form with the microsomes. Digitonin treatment of cells causes rapid release into the medium of the cytosolic, but not the microsomal form of the cytidylyltransferase. Incubation of hepatocytes for 10 min with phospholipase A2 (0.9 units/dish) in the medium, resulted in a 33% decrease in the cytidylyltransferase activity released by digitonin treatment (2.5 +/- 0.15 nmol/min per mg compared to 3.9 +/- 0.10 nmol/min per mg in the control). In agreement with the digitonin experiments, incubation with 0.9 units/dish of phospholipase A2 resulted in a decrease in the cytidylyltransferase activity in the cytosol (from 4.3 +/- 0.10 nmol/min per mg to 2.6 +/- 0.14 nmol/min per mg) and a corresponding increase in the microsomal fraction (from 0.9 +/- 0.16 nmol/min per mg to 1.8 +/- 0.20 nmol/min per mg). The effect of phospholipase A2 on cytidylyltransferase translocation was concentration- and time-dependent. Incubation of hepatocytes in the presence of phospholipase A2 (0.9 units/dish) for 10 min prior to pulse-chase experiments resulted in an increase in radiolabel incorporation into phosphatidylcholine (from 2.4 +/- 0.02.10(-5) dpm/dish to 3.1 +/- 0.1.10(-5) dpm/dish) and a corresponding decrease in radiolabel associated with the choline (from 2.5 +/- 0.05.10(-5) to 1.4 +/- 0.03.10(-5) dpm) and phosphocholine fractions (from 8.5 +/- 0.07.10(-5) to 6.9 +/- 0.05.10(-5) dpm). We conclude that phospholipase A2 can cause a stimulation of CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in cultured rat hepatocytes.     

Purification of branched chain aminotransferase from rat heart mitochondria

This paper presents the first purification of the branched chain aminotransferase (EC 2.6.1.42) from rat heart mitochondria. The enzyme has been purified from the 100,000 x g supernatant obtained after sonication and ultracentrifugation of rat heart mitochondria. A combination of open column chromatography, high pressure liquid chromatography (HPLC), and discontinuous polyacrylamide disc gel electrophoresis was used. The key step in the procedure was hydrophobic interaction chromatography on HPLC. The final purification step was polyacrylamide disc gel electrophoresis where the enzyme appeared as a doublet. When electroeluted from the gel, each of these bands had the same specific activity demonstrating that there are two forms of the purified enzyme which differ slightly in electrical charge. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these two enzyme forms appeared as a single band with a molecular mass of 43 kDa. Size exclusion chromatography on Sephacryl S-100 identified the enzyme as a 50-kDa protein. These experiments argue against the existence of a dimeric form of this enzyme. The ratio of enzyme activity with leucine (0.84), valine (0.88), or glutamate (0.66) as amino acid substrate versus isoleucine remained constant throughout the purification procedure. Specific activity of the final preparation was 66 units/mg of enzyme protein. Polyclonal antibodies against the purified enzyme were raised in rabbits. On an immunoblot the antiserum recognized a 43-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate but did not recognize any proteins in rat brain cytosol. Quantitative immunodot assay resulted in an estimated enzyme content of about 100 micrograms of branched chain aminotransferase protein/g of heart, wet weight. Finally, 97% of the heart branched chain aminotransferase activity could be neutralized by the antiserum, but the antiserum would not neutralize aminotransferase activity in brain cytosol. These data suggest that close sequence homology does not exist between the two proteins.     

Purification of Clostridium toxoids.

A two-step fractionation procedure was applied for purification and concentration of the individual Clostridium toxoids. The toxoids were precipitated with hydrochloric acid in the presence of sodium sextametaphosphate, then antigenic fractions were separated from inactive contaminants by Sephadex G-75 filtration. Specific activity of the preparations thus obtained, as determined by Mancini radial immunodiffusion, was 150--565 binding units per mg of protein nitrogen for Clostridium perfringens toxoid, 204--352 binding units for Clostridium oedematiens toxoid and 26.6 -- 51.2 binding units for Clostridium septicum toxoid.     

Detection of endothelial nitric oxide release with the 2,3-diaminonapthalene assay

The reliable measurement of nitric oxide (NO) production by endothelial cells in vitro has become an important tool for investigating mechanisms of endothelial dysfunction. This study evaluates measuring NO production by cultured porcine pulmonary artery endothelial cells (PAEC) using the assay based on the fluorometric detection of 1-(H)-naphthotriazole, the fluorescent product of the reaction between nitrite (NO2-) and 2,3-diaminonapthalene (DAN). To stimulate NO production, PAEC were treated for 60 min with agonists known to stimulate endothelial NO production. The DAN assay was unable to detect NO production from agonist-stimulated PAEC. In contrast, chemiluminescence analysis, which detects NO, NO2-, and nitrate (NO3-) (collectively referred to as NO(x)), detected significant increases in NO(x) from stimulated PAEC. Nitrate reductase-mediated reduction of NO3-to NO2- in media from stimulated PAEC enhanced the ability of the DAN assay to detect NO release from PAEC. These results provide the first direct comparison of the sensitivity of these two commonly employed assays. Our findings emphasize that NO3-reduction may be required to enable the DAN assay to detect small amounts of NO produced by cultured endothelial cells.