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J. Biol. Chem., Vol. 276, Issue 32, 30085-30091, August 10, 2001

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Distinction between Nitrosating Mechanisms within Human Cells and Aqueous Solution^*

Michael Graham Espey, Katrina M. Miranda, Douglas D. Thomas, and David A. Wink

From the Radiation Biology Branch, Division of Clinical Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, February 23, 2001, and in revised form, June 7, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The quintessential nitrosating species produced during NO autoxidation is N₂O₃. Nitrosation of amine, thiol, and hydroxyl residues can modulate critical cell functions. The biological mechanisms that control reactivity of nitrogen oxide species formed during autoxidation of nano- to micromolar levels of NO were examined using the synthetic donor NaEt₂NN(O)NO (DEA/NO), human tumor cells, and 4,5-diaminofluorescein (DAF). Both the disappearance of NO and formation of nitrosated product from DAF in aerobic aqueous buffer followed second order processes; however, consumption of NO and nitrosation within intact cells were exponential. An optimal ratio of DEA/NO and 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) was used to form N₂O₃ through the intermediacy of NO₂. This route was found to be most reflective of the nitrosative mechanism within intact cells and was distinct from the process that occurred during autoxidation of NO in aqueous media. Manipulation of the endogenous scavengers ascorbate and glutathione indicated that the location, affinity, and concentration of these substances were key determinants in dictating nitrosative susceptibility of molecular targets. Taken together, these findings suggest that the functional effects of nitrosation may be organized to occur within discrete domains or compartments. Nitrosative stress may develop when scavengers are depleted and this architecture becomes compromised. Although NO₂ was not a component of aqueous NO autoxidation, the results suggest that the intermediacy of this species may be a significant factor in the advent of either nitrosation or oxidation chemistry in biological systems.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

An early discovery in the nitrogen oxide field was that macrophages utilize nitric oxide (NO) derived from L-arginine in their tumoricidal armature (1). Although NO directly reacts primarily with metalloproteins, reactive nitrogen oxide species (RNOS)¹ formed during the autoxidation of NO can engage an alternate and broader range of molecular targets (2-5). The salient nitrosating species produced during NO autoxidation is N₂O₃, which can react with amines, thiols, or hydroxyl groups to form NO adducts (6). Nitrosative mechanisms have been implicated in ion conductance (7-10), signal transduction (11), glycolysis (12), apoptosis (13), and DNA repair (14, 15), underscoring the pivotal role these modifications may play in many facets of cellular function. Excessive formation of N₂O₃ can exert toxicological changes that culminate as nitrosative stress if critical systems become adversely affected (5, 16, 17). Therefore, the chemistry leading to N₂O₃ formation may figure prominently in both functional and cytotoxic aspects of NO in vivo.

Several mechanisms have been proposed for autoxidation of NO (17-30). However, few studies have examined this process in the context of the intracellular milieu (29, 30). We tested the hypothesis that the autoxidation process within the architecture of intact cells is distinct from that which occurs in the aqueous extracellular medium. The results suggest that NO₂ is not an intermediate species during NO autoxidation in aqueous phase. Cellular hydrophobic domains in conjunction with scavenger composition and location may serve to focus nitrosation chemistry to discrete sites during NO autoxidation in biological systems.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Compounds Used to Generate RNOS-- NaEt₂NN(O)NO (DEA/NO) was a generous gift from Dr. Joseph Saavedra (NCI, National Institutes of Health, Frederick, MD). DEA/NO decomposes to release free NO into solution with a half-life of 2.5 min at neutral pH and 37 °C (Refs. 31 and 32; see also Fig. 2A). Stock solutions (~1 mM) were prepared in 10 mM NaOH and were stored at 20 °C, and concentrations were determined from the absorbance values at 250 nm in 10 mM NaOH ( = 8000 M¹ cm¹; Ref. 31) directly prior to use. The rate of DEA/NO decomposition was unaltered by the presence of either DAF or 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO).

Ascorbic acid, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), buthionine sulfoximine (BSO), diethylenetriaminepentaacetic acid (DTPA), and glutathione (GSH) were purchased from Sigma. PTIO, 4,5-diaminofluorescein-2 (DAF), and DAF-diacetate were purchased from Calbiochem (San Diego, CA).

Initial experiments indicated that, during aerobic decomposition of 0.5 µM DEA/NO in a PBS-buffered solution, the fluorescence signal from the DAF reaction product triazolofluorescein was linearly dependent on DAF, with concentrations ranging from 1 nM to 1 µM. At higher DAF concentrations, nonlinearity in fluorescence response was observed indicative of an interference effect; therefore, solution data were accumulated in PBS solutions containing 1 µM DAF and the metal chelator DTPA (50 µM) unless otherwise indicated. The level of ambient light was kept to a minimum during all steps involving DAF. For anaerobic analysis, the assay buffer was deaerated by bubbling with argon through a septum-sealed cuvette.

Cell Conditions-- The human cancer cell lines MCF-7 (breast), A-549 (lung), and HT-29 (colon) were obtained from American Type Tissue Collection (Manassas, VA) and were cultured as attached cells to 80% confluence in either T-75 flasks (Falcon) or 96-well, black-walled plates (Corning) containing RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (HyClone Laboratories, Logan, UT), 4.5 g/liter glucose, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified incubator with 5% CO₂ and 95% air. As indicated, cells were treated with either 50 µM BSO or 200 µM ascorbate for 16 h prior to testing. Cells subsequently were gently rinsed with PBS and incubated at 37 °C for 30 min with a PBS solution containing 5 µM DAF-diacetate and DTPA (50 µM). After replacement of the DAF solution with fresh PBS, the cells were dislodged and were either washed three times by a cycle of suspension and centrifugation or, in the case of the 96-well plate format, were rinsed by cycles of aspiration and PBS addition.

Differences in uptake efficiency of DAF between tumor cell lines were determined by reacting lysates (two pulses of 10 s with an ultrasonicator; Cole-Palmer, Vernon Hills, IL) of each sample preparation with a large excess of DEA/NO (500 µM) to give maximal fluorescent signal. The total DAF uptake by MCF-7 and A-549 cells was equivalent, whereas that by HT-29 cells was ~65-75% lower. This approach also confirmed that the total level of intracellular DAF for each cell line was in excess relative to the concentration of introduced nitrogen oxides in subsequent experiments. Ultrasonication of intact cells following complete decomposition of DEA/NO did not affect the intensity of fluorescence, indicating that interference quenching within the enclosure of the cell did not occur. Treatment with BSO augmented the absolute level of intracellular DAF roughly 3-4-fold in MCF-7 and A-549 cells, but had little effect on DAF uptake in HT-29 cells. Pre-incubation of MCF-7, A-549, and HT-29 cells with ascorbate did not significantly alter DAF uptake. The diacetate derivative of DAF was designed to aid in retention of DAF within intact cells. The level of DAF leakage from cells 30 min after suspension in fresh PBS-buffered solution was determined from the signals obtained in supernatant relative to the lysed cell pellet following exposure to DEA/NO (500 µM). Leakage of the dye back into solution or incidental cellular lysis resulted in a signal that was 2-3% of the total amount of fluorophore present in 1 × 10⁶ cells. To avoid misinterpretation of this solution artifact as intracellular signal, DAF-loaded cells were washed and suspended into fresh buffer immediately prior to testing. Trypan blue dye exclusion indicated that viability of intact cells at the end of all treatment conditions was in the range of 92-100%.

Instrumentation and Data Analysis-- UV-visible spectroscopy was performed with a Hewlett-Packard 8452A diode array spectrophotometer. Fluorescence measurements were obtained on a PerkinElmer Life Sciences LS50B fluorometer with excitation at 495 and emission at 515 nm with either 2.5- or 5.0-mm slit widths as indicated. The reaction solution (2 ml) was stirred and maintained at 37 °C with a water-jacketed cuvette holder. Additional fluorescence measurements were obtained on a PerkinElmer Life Sciences HTS 7100 fluorescent plate reader (200-µl volume, 37 °C). Kinetic analyses (KaleidaGraph software; Synergy, Reading, PA) of fluorescence signal increases were performed on data sets excluding the first 2 min to ignore the contribution from DEA/NO decay prior to the NO maximum. Kinetic simulations were obtained using Stella II software (High Performance Systems). NO measurements were made using a NO-specific electrochemical probe (World Precision Instruments, Sarasota, FL) suspended into a fluorometer cuvette controlled by a DUO18 amplifier and software. Signals were calibrated using argon-purged PBS solutions of saturated NO gas (Matheson, Montgomeryville, PA) following determination of concentration with ABTS (660 nm,  = 12,000 M¹ cm¹; Ref. 33). The background signals were assessed using nitrite or DEA/NO pre-incubated for 4 h in buffer. The microscopic characteristics of DAF-loaded cells were viewed using a Zeiss LSM 210 microscope equipped with a mercury lamp and a fluorescein filter set.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

N-Nitrosation of the aromatic vicinal amines of DAF results in formation of a triazolofluorescein product that exhibits fluorescence with a high extinction coefficient and quantum yield (34). Decomposition of DEA/NO in a PBS-buffered solution containing DAF generated fluorescent product (Fig. 1A). Since NO was introduced by decay of DEA/NO (k = 4.6 × 10³ s¹; Ref. 30) as opposed to bolus addition, the temporal increase in signal intensity is reflective initially of NO release from this donor compound (Reaction 1).

As NO levels decreased with decay of DEA/NO, the rate-limiting step for fluorescent product formation becomes autoxidation, which is second order in NO (Reaction 2; k = 8 × 10⁶ M² s¹; Refs. 18 and 19).

Kinetic analysis of the data, excluding the first 2 min, confirmed that the signal increase conformed to an apparent second order process (Fig. 1A, k = 4.8 ± 0.4 × 10³ M¹ s¹). Electrochemical detection of the NO concentration in solution corroborated this interpretation as the free NO level rose to a maximum, indicative of production exceeding the rate of consumption, then declined by a second order process (Fig. 2A, k = 1.6 ± 0.1 × 10³ M¹ s¹, buffer). At the detection limits of the fluorometer, the process by which low nanomolar levels of NO elicited increases in fluorescent product remained second order (data not shown). Inclusion of the metal chelator DTPA did not affect the reaction. Addition of nitrite, diethylamine, or previously decomposed DEA/NO did not generate fluorescent product when DAF was present in either solution or within cells. DAF nitrosation during decomposition of DEA/NO was dependent on O₂ and did not develop upon re-oxygenation of solutions previously reacted in the absence of O₂. These results indicated that NO does not nitrosate DAF directly and were consistent with formation of N₂O₃ consequent to autoxidation of NO.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Fluorescence changes elicited by DEA/NO decomposition with DAF present free in solution and within either intact or lysed MCF-7 tumor cells. A, DEA/NO (1 µM) was added to a PBS-buffered solution (2 ml, stirring, 37 °C) containing DAF (1 µM) and the metal chelator DTPA (50 µM). The rate of increase in fluorescent product was second order (k = 4.8 ×103 M1 s1, line overlay). B, DAF-diacetate was incorporated into cells during a 30-min incubation period followed by centrifugation and wash steps to remove extracellular DAF. Cells (2 × 106) were resuspended in buffer as in A and remained either intact or were lysed by a brief sonication pulse. Increase in fluorescence after addition of DEA/NO (10 µM) followed a single exponential with intact cells (k = 3.8 × 103 s1, line overlay), while the rate law in lysate was second order (k = 5.2 ×103 M1 s1, line overlay). C, DEA/NO (0.5 µM) in either the presence or absence of PTIO (5 µM) was added to DAF in solution as in A. The reaction was first order in the presence of PTIO (k = 5.5 × 103 s1, line overlay) and remained second order in its absence (k = 4.4 × 103 M1 s1, line overlay). D, DEA/NO (2.5 µM) was added to buffer containing PTIO (25 µM) and either intact or lysed cells as in B. Fluorescent product formation was first order under both conditions (k = 9.3 × 103 s1 and k = 8.2 × 103 s1, respectively, line overlays). Fluorescence changes were monitored at ex/em of 495/515 nm with either 5-mm (A and B) or 2.5-mm (C and D) slit widths.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Comparison of NO disappearance in the presence and absence of MCF-7 tumor cells. A, signals were obtained from an electrode designed for an enhanced selectivity to NO (high oxidation potential, anion resistant coating). Representative responses (µM NO) observed following addition of DEA/NO (1 µM) to either PBS-buffered solution (2 ml, 37 °C, stirring) or buffer containing 1 × 106 MCF-7 tumor cells are shown. B, simulated data showing the disappearance of NO and nitrosated formation under conditions where the rate of NO autoxidation exceeds the rate of DEA/NO decomposition (4.6 × 103 s1) at all time points (consumption) and where cellular consumption (2 × 103 s1) competes with autoxidation (acceleration).

To test the hypothesis that the intracellular milieu alters nitrosation, tumor cells were loaded with DAF and remained either intact or were ruptured by ultrasonication immediately prior to exposure to equal concentrations of DEA/NO. The fluorescent signal generated from N₂O₃ reaction with DAF inside intact MCF-7 cells was 54 ± 12% lower than the signal observed with corresponding lysate (Fig. 1B). Comparable results were obtained with A-549 cells (data not shown). Similar to cell-free solution, the rate of fluorescence increase following cellular disruption was second order (Fig. 1B, lysed, k = 5.2 ± 0.8 × 10³ M¹ s¹). In contrast, the appearance of fluorescent product from DAF localized within intact cells was exponential with an apparent first order rate constant of 4.0 ± 0.3 × 10³ s¹. Given the similarity between this value and the rate for DEA/NO decomposition (4.6 × 10³ s¹; Ref. 32), we tested the hypothesis that the enhanced solubility of NO and O₂ within the hydrophobic phase of cellular membranes may accelerate the rate of N₂O₃ formation (29) beyond the rate of DEA/NO decomposition at all time points. Simulations predicted that the maximum NO concentration would decrease 4-fold under these conditions (Fig. 2B); however, this model was incongruous with experimental data. The presence of MCF-7 tumor cells (1 × 10⁶, 2-ml volume, stirring, 37 °C) resulted in only a small reduction (~20%) in the electrochemical signal peak height during decomposition of 1 µM DEA/NO (Fig. 2A). If cells consume NO (30), then nitrosation during autoxidation would be decreased. An increased first order rate of NO disappearance (k = 2.5 ± 0.4 × 10³ s¹) was evident from solution containing cells exemplified by a faster return of the electrochemical signal to base line (Fig. 2A). Consistent with this, inhibition of fluorescence generated with DEA/NO and free DAF in aerobic solution was observed upon addition of intact tumor cells (Fig. 3A). Notably, a reciprocal plot of these data revealed that inhibition occurred by two distinct processes (Fig. 3B).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Characteristics of extracellular DAF nitrosation inhibition by MCF-7 tumor cells. DEA/NO (1 µM) was added to 200 µl of PBS-buffered solution containing free DAF (1 µM), DTPA (50 µM), and the indicated number of intact unlabeled MCF-7 tumor cells in suspension with (C) or without (A) PTIO (10 µM). Fluorescence was measured (ex/em 490/525 nm) after a 45-min incubation period at 37 °C (unstirred). B, a reciprocal plot of fluorescence intensity in A revealed that the data fit to two separate regression lines (m = 0.028 and 0.0018). D, a reciprocal plot of data from C yielded a single regression line with a slope (0.0031) similar to the slower process in B. Representative data are given as the mean ± S.E. of triplicate wells (n = 3). Note that the scale of the y axis in panel C is 10-fold greater than that in A.

PTIO is an imidazolineoxyl N-oxide spin-trap that converts NO directly to NO₂ in an oxygen transfer reaction (k = 1 × 10⁴ M¹ s¹; Refs. 35 and 36; Reaction 3).

We used PTIO as a tool to investigate the relationship between cells and NO₂ formation during NO autoxidation. Electrochemical measurements verified that the level of free NO present in solution during DEA/NO decomposition was decreased by PTIO in a dose-dependent fashion (data not shown). PTIO at 500-fold or greater excess relative to DEA/NO resulted in nearly complete inhibition of fluorescent product formation from DAF (Fig. 4A). Under these conditions, conversion of NO to NO₂ by PTIO approached 100%, indicating that NO₂ alone cannot nitrosate DAF. In contrast, the rate and yield of product formation in both cells (Fig. 1C) and solution (Fig. 4A) were markedly augmented with lower concentrations of PTIO, where some portions of NO remained available to react with NO₂, forming N₂O₃ (Reaction 4; k = 1.1 × 10⁹ s¹, Refs. 22 and 26).

The optimal ratio to achieve maximal nitrosation of DAF was ~10-15-fold excess PTIO to DEA/NO (Fig. 4A). The fluorescent product yield under these conditions was 10-fold higher relative to the signal observed with autoxidation alone (Fig. 1C). The absence of a headspace in the reaction vessel had a negligible effect (±5%; data not shown) on fluorescent product formation during DEA/NO decomposition in aerobic solution. Therefore, the marked increase in nitrosated product yield observed with NO/PTIO was not the result of NO capture prior to escape into room air. A double-reciprocal plot of fluorescence as function of DAF concentration indicated there was a vast difference between the affinities of the nitrosating species produced during DEA/NO decomposition in either the presence (1/X_int = 0.44 ± 0.04 µM) or absence (1/X_int = 5.5 ± 0.6 µM) of PTIO (Fig. 4B). At infinite concentrations of DAF, the maximal fluorescence values were similar (1/Y_int = 2900, DEA/NO; 4000, DEA/NO + PTIO), indicating that the absolute levels of nitrosating species formed were comparable.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Relationship between PTIO and DEA/NO concentrations on formation of fluorescence from DAF. A, DEA/NO (1 µM) was decomposed in aerobic PBS-buffered solution containing DAF (1 µM), DTPA (50 µM), and PTIO as indicated. The level of fluorescence was measured (ex/em 490/525 nm) after a 45-min incubation period at 37 °C (200-µl wells, unstirred). B, double-reciprocal plots of fluorescence (collected at ex/em: 495/515 nm, slit widths 2.5 mm) versus DAF concentration. The relative affinities of the nitrosating species formed by DEA/NO (1 µM) decomposition in aerobic PBS-buffered solution containing DTPA (50 µM) and DAF (1 nM to 1 µM) for 1 h at 37 °C in either the absence (m = 0.00186, b = 0.00034, r = 0.997) or presence (m = 0.00011, b = 0.00025, r = 0.999) of PTIO (10 µM). Values shown are the mean ± S.E. of triplicate wells from representative data (n = 3).

Although PTIO is cell-impermeable, the NO/PTIO combination resulted in an augmented level of intracellular DAF nitrosation relative to that achieved solely with NO autoxidation (Fig. 1D). Fluorescent production formation was further enhanced in lysate relative to the signal obtained with DAF inside intact cells. Experiments with PTIO, DEA/NO, and free DAF in solution showed that addition of intact cells progressively inhibited formation of nitrosated product (Fig. 3C). A reciprocal plot of these data indicated a single inhibitory process (Fig. 3D). The slope (m = 0.0031) of this line was similar to the slower process (m = 0.0018) observed with DEA/NO in the absence of PTIO (Fig. 3B).

The role of endogenous quenchers of nitrosation was evaluated. Nitrosation of DAF (1 µM) in aqueous buffer was more susceptible to ascorbate (IC₅₀ = 2 µM, r² = 0.996) than GSH (IC₅₀ = 90 µM, r² = 0.990) during DEA/NO decomposition (1 µM). Extracellular GSH at a ratio of 200:1 completely inhibited intracellular nitrosation of DAF during DEA/NO decomposition. In contrast, much lower levels of GSH (~6:1) quenched DAF product formation within cells when nitrosation was elicited in the presence of PTIO (Fig. 5). Cells were incubated with either ascorbate or BSO, the competitive inhibitor of -glutamylcysteine synthetase (37, 38), to determine the degree to which ascorbate or GSH may protect intracellular constituents from nitrosation. The level of fluorescence generated by exposure to DEA/NO was increased roughly 2-fold in MCF-7 and A-549 cells depleted of GSH with BSO (Fig. 6). Nitrosation in GSH-depleted HT-29 cells was relatively unchanged (data not shown). The rate constants for fluorescent product formation in MCF-7 cells treated with BSO were first order (4.0 ± 0.8 × 10³ s¹), similar to those observed in non-BSO-treated cells (data not shown). Fluorescence maxima produced by DEA/NO were reduced ~30-60% in each tumor cell type previously supplemented with ascorbate (Fig. 5).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of GSH on fluorescence changes elicited by DEA/NO ± PTIO with DAF within MCF-7 tumor cells. DAF-diacetate was incorporated into cells during a 30-min incubation period. Following centrifugation and wash steps to remove extracellular DAF, cells (2 × 106) were resuspended in PBS-buffered solution (2 ml, stirring, 37 °C) containing DTPA (50 µM) either without (A) or with (B) PTIO (25 µM) and GSH (30 µM) as indicated. Increases in fluorescence after addition of DEA/NO (5 µM) were monitored at ex/em of 495/515 nm with 2.5-mm slit widths.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Influence of intracellular ascorbate and GSH levels on nitrogen oxide-induced fluorescence in tumor cells. The human tumor cell lines MCF-7 or A-549 were plated into a 96-well culture plate at a density of 50 × 105 and were treated for 16 h with either ascorbate (Asc, 200 µM) or BSO (5 mM). Following a 1-h incubation period in PBS-buffered solution containing 5 µM DAF-diacetate, cells were gently rinsed twice and reacted with DEA/NO (20 µM) in buffer for 1 h at 37 °C. Representative data are shown as the mean ± S.E. of quadruplicate wells (n = 3).

A heterogeneous pattern of intracellular fluorescence was evident when tumor cells loaded with DAF were viewed under a microscopic following exposure to DEA/NO (data not shown). Qualitatively, fluorescence was often organized into discrete regions resulting in punctate appearance. A diffuse pattern that filled the entire cell, however, was evident in many cases. The fluorescence per individual cell ranged from none (background) to greater than 95%.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We tested the hypothesis that the chemistry of NO autoxidation in aqueous solution was not synonymous with the reaction within intact cells. Nitrosation of free DAF in aerobic solution occurred by an apparent second order process consistent with disappearance of NO as the rate-limiting step (18, 19) (Reaction 2 and Fig. 1A). The temporal increase in fluorescent signal elicited by DEA/NO decomposition appeared to be first order when DAF was intracellular (Fig. 1B). The higher solubility of both NO and O₂ within cellular membranes accelerates the rate of NO autoxidation, but does not significantly alter the second order rate constant for N₂O₃ formation (29). Simulations of NO disappearance by hydrophobic solubility affects alone were inconsistent with experimental results (Fig. 2, A and B). In agreement with our findings (Fig. 2A), cellular consumption of NO by a non-nitrosating pathway with a modeled rate constant of 2 × 10³ s¹ predicted a lower yield of nitrosated product and accelerated NO depletion. Autoxidation may predominate at high concentrations of NO (second order in NO) until levels fall to a point where cellular consumption becomes competitive. This threshold phenomenon cloaks the overall appearance of fluorescent product within cells as an apparent first order process.

Dynamic modulation of both the rates of NO formation by NOS isoforms (2, 16, 39, 40) and of NO consumption by different cell types (30) may serve to regulate nitrosation chemistry within a particular region of tissue. NO levels generated from constitutive NOS isoforms are generally limited by feedback inhibition from nitrosyl complex formation at the catalytic heme site (39, 40). Several studies have noted nitrosation generated within neurons and endothelia using DAF (41-43), while we observed second order dependence for fluorescent product formation in solution at submicromolar concentrations of NO from synthetic donors (e.g. Fig. 1C). These data are consistent with the viewpoint that physiologic NO formation can lead to subtle levels of nitrosation, which may function to modulate residues critical for channel gating, subunit dimerization, and enzyme activity. Measurements in the current in vitro studies were conducted in the absence of glucose. We have observed that manipulation of mitochondrial status in MCF-7 cells (± glucose, rotenone, actinomycin D) had little effect on both NO consumption and extracellular DAF nitrosation. However, intracellular DAF nitrosation levels were influenced by respiration, albeit only modestly (10-15%; data not shown). Localization of DAF within mitochondria has been examined (44), and intraorganelle nitrosation mechanisms remain an active area of inquiry.

Although cells can affect the rate of nitrosation, nucleophilic substances such as GSH and ascorbate can limit the yields of nitrosation by competitive scavenging of N₂O₃ (16, 19, 45-48). MCF-7 breast carcinoma cells contain an estimated 5-10 mM intracellular GSH (38), a concentration that completely quenches autoxidation-elicited nitrosation in solution by even supraphysiologic NO levels. Despite this, intracellular nitrosation of DAF was elicited with submicromolar NO. Moreover, the approximate 2-fold increases in DAF nitrosation in BSO-treated cells were similar to the increases in fluorescence intensity observed by diluting the intracellular contents upon lysis (Fig. 1B). Conversely, enrichment of intracellular ascorbate levels inhibited intracellular nitrosation from NO autoxidation (Fig. 6). The results show that the scope of protection by GSH and ascorbate are dependent on both their differential affinity and concentration. Although chronic nitrosative stress globally depletes these defenses, salubrious nitrosative reactions that function as redox switches may be specifically sheltered from quenching in compartments that exclude these agents (e.g. channels, vacuoles). Caution must be taken when examining modulation of intracellular protein function by nitrosative mechanisms outside the context of the intact cells in tissue or biological fluids (e.g. purified recombinant protein in simple buffer), which may result in a misinterpretation of the actual susceptibility to nitrosative stress in vivo (49, 50).

To test whether the intermediates of the NO autoxidation reaction in water produces intermediates different from those formed in the gas phase/hydrophobic media (18, 19, 27, 28), we formed NO₂ from the reaction between NO and PTIO (Reactions 3 and 4; Refs. 35 and 36). In addition to circumventing the rate-limiting step in autoxidation, the optimal balance of PTIO and DEA/NO dramatically augmented the rate of DAF nitrosation and markedly increased product yield (Figs. 1C and 4A). A double-reciprocal plot of fluorescence versus DAF concentration demonstrated that the nitrosating species formed by the NO/PTIO route has a much higher affinity for DAF than that formed during NO autoxidation in aqueous media (Fig. 4B). For both DEA/NO and DEA/NO + PTIO paradigms, the end point product yield at saturating concentrations of DAF (1/Y_int) were similar. These data indicate that the absolute concentrations of nitrosating species formed by either pathway are comparable. Thus, the striking difference in the affinity of these species was likely due to their selectivity toward DAF. Differences in affinity were not restricted to DAF, but were also observed with biological substrates such as GSH (Fig. 5) as well as ABTS and ferrocyanide (data not shown). Consistent with these data, the reactivity of gaseous N₂O₃ dissolved in water was observed to be remarkably different than N₂O₃ formed from acidic nitrite (28). Taken together, these results raise the possibility that distinct nitrosating intermediates may exist in biological systems (18, 19, 27, 28, 51, 52). The low affinity profile observed during NO autoxidation in buffer calls into question the proposed mechanism involving NO₂ intermediacy.

Analysis of fluorescence from DAF free in aqueous solution as a function of intact cell number during DEA/NO decomposition revealed two processes for inhibition of nitrosation (Fig. 3, A and B). The initial process was consistent with NO consumption by intact cells. The slope of the second process was similar to the inhibition profile observed with NO + NO₂ generated artificially with DEA/NO and PTIO (Fig. 3, C and D). Based on this similarity, we propose that cells provide an environment, possibly the hydrophobic portion of membranes, which leads to formation of a high affinity nitrosating species. High levels of extracellular GSH were able to inhibit nitrosation of intracellular DAF (Fig. 5). These data suggest the existence of a species with sufficient lifetime to diffuse from the cellular membranes into the medium. Based on rates of hydrolysis, the most likely candidate is NO₂, which could combine with NO resulting in formation of N₂O₃ (Reaction 4).

These findings raise the possibility that distinct profiles of NO autoxidation may exist within cells versus the aqueous interstitium. This dichotomy would be of consequence due to the differential reactivity of N₂O₃ formed by each pathway and may serve to focus chemical modifications during NO diffusion into discrete subcellular domains at both the tissue and molecular levels. Critical to this balance is the fate of NO₂, which may lead to nitrosative, oxidative, or nitrative reactions depending on the levels of appropriate substrates and the concentration of NO. An imbalance in the levels of key endogenous scavenger substances for RNOS can lead to either nitrosative or oxidative stress. These results provide a novel framework for the mechanisms of nitrosation at physiological NO concentrations in biological systems.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Radiation Biology Branch, NCI, National Institutes of Health, Bldg. 10, Rm. B3-B69, Bethesda, MD 20892. Tel.: 301-496-7511; Fax: 301-480-2238; E-mail: sp@nih.gov.

Published, JBC Papers in Press, June 12, 2001, DOI 10.1074/jbc.M101723200

	ABBREVIATIONS

The abbreviations used are: RNOS, reactive nitrogen oxide species; DEA/NO, NaEt₂NN(O)NO; DAF, 4,5-diaminofluorescein-2; PBS, phosphate-buffered saline; DTPA, diethylenetriaminepentaacetic acid; PTIO, 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide; BSO, buthionine sulfoximine; ABTS, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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