©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetic Analysis of the Fate of Nitric Oxide Synthesized by Macrophages in Vitro(*)

(Received for publication, February 2, 1995; and in revised form, September 15, 1995)

Randy S. Lewis (1)(§) Snait Tamir (2) Steven R. Tannenbaum (2) William M. Deen (1)(¶)

From the  (1)Department of Chemical Engineering, the (2)Department of Chemistry, and the (3)Division of Toxicology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To investigate the fate of nitric oxide (NO) synthesized by activated macrophages, the concentrations of NO and its principal reaction products, nitrite (NO(2)) and nitrate (NO(3)), were measured as a function of time in suspension cultures of RAW264.7 macrophages attached to microcarrier beads. Synthesis of NO became evident 2-5 h after stimulation of the cells, and steady concentrations of NO were achieved after about 9 h. The appearance of NO in the extracellular fluid coincided with the appearance of NO(2) and NO(3), which were formed thereafter at approximately equal and constant rates. Using a kinetic model based on rate constants measured previously in cell-free systems, only half of the NO(2) formed could be accounted for by the reaction of NO with O(2). It is known that NO reacts with superoxide (O(2)) to give peroxynitrite and that NO also reacts with peroxynitrite to yield NO(2), so that the latter reaction may explain the ``excess'' NO(2) formation. Adding superoxide dismutase to the medium markedly reduced the ratio of NO(3) to NO(2), consistent with the hypothesis that NO(3) in the medium results primarily from the extracellular reaction of NO with O(2). The addition of morpholine, a model amine, resulted in formation of N-nitrosomorpholine, concurrent with the other products. Measured rates of nitrosomorpholine formation were 6-fold lower than predictions based on kinetics in simple solutions, suggesting that in the cell culture system there were additional reactions that lowered the concentration of nitrous anhydride, the principal nitrosating agent formed from NO and O(2).


INTRODUCTION

Nitric oxide (NO) is a biological messenger synthesized by a wide variety of cells, including macrophages, endothelial cells, neutrophils, neurons, and hepatocytes. It is involved in blood pressure regulation, inhibition of platelet aggregation, and neurotransmission, among other functions(1) . In addition, when present at sufficient concentrations, NO and its nitrogen oxide derivatives have cytotoxic and mutagenic effects. Nitrous anhydride (N(2)O(3)), an intermediate in the reaction of NO with molecular oxygen, may damage DNA through direct nitrosation of primary amines on DNA bases. A more indirect pathway for DNA damage involves metabolic products of N-nitrosamines, which are formed by the reaction of N(2)O(3) with various secondary amines(2) . Cytotoxic and mutagenic effects may result also from reactions involving NO and superoxide (O(2)), such as lipid peroxidation via peroxynitrite (ONOO), an intermediate in the reaction of NO with O(2)(3) .

Although there has been much interest in the biological effects of NO and species formed by the reactions of NO with O(2) and O(2), a quantitative understanding of the products of these reactions has been difficult to achieve. The difficulty results from the complex chemistry of nitrogen oxides and, in some cases, the extremely low concentrations of the species involved. Many cells, such as activated macrophages, synthesize both NO and O(2), resulting in the formation of NO(2) and NO(3) as end products in various ratios (4, 5, 6, 7, 8) . Intermediates such as N(2)O(3) and ONOO are not directly measurable in cell cultures. An important component of efforts to assess the effects of NO-generating cells on neighboring cells or tissues is the characterization of the extracellular reaction kinetics. An accurate kinetic model would offer insight into the observed amounts of the end products, as well as the probable levels of the trace intermediates.

The experiments reported here were designed to test the extent to which a kinetic model developed recently for cell-free systems (9) can explain the observed extracellular concentrations of NO, nitrite (NO(2)), nitrate (NO(3)), and N-nitrosomorpholine (NMor) (^1)in the presence of activated macrophages in culture. Of importance in the experimental design was the attachment of the macrophages to microcarrier beads. The use of a suspension culture allowed for a well mixed aqueous solution, such as would not necessarily be the case with cell culture plates. Macrophages were stimulated with Escherichia coli lipopolysaccharide and -interferon to generate NO, and the concentrations of NO, NO(2), NO(3), and NMor were measured as functions of time. It has been shown previously that the model amine chosen, morpholine, is nitrosated in the presence of activated macrophages to form NMor(4, 10) , most likely by its reaction with N(2)O(3)(9, 10) . Accordingly, the rate of formation of NMor was used to indirectly assess the concentration of N(2)O(3). Superoxide dismutase (SOD), which scavenges O(2), was added to some of the macrophage cultures. In oxygenated buffer solutions NO(3) is not formed from NO in the absence of O(2)(11) , and this is true also for cell culture medium. Thus, the effect of SOD on the relative rates of NO(2) and NO(3) formation provides an indication of the levels of O(2) present in the extracellular solution.


MATERIALS AND METHODS

Reagents

Auto-POW-modified Eagle's medium without phenol red, L-glutamine, or NaHCO(3) was purchased from ICN Biomedicals (Costa Mesa, CA), and NaHCO(3) was obtained from Mallinckrodt (Paris, KY). Dulbecco's modified Eagle's medium, sodium pyruvate, L-glutamine, heat-inactivated calf serum (HICS), and modified Eagle's medium essential amino acids were obtained from BioWhittaker (Walkersville, MD). Trypan blue, HEPES, D-glucose, E. coli lipopolysaccharide (LPS) (serotype 0127:B), and modified Eagle's medium vitamins were obtained from Sigma. Mouse -interferon was obtained from Genzyme (Cambridge, MA) or Boehringer Mannheim. Bovine erythrocyte SOD (5000 units/mg, M(r) = 32,500) was from Boehringer Mannheim, and morpholine (Mor) was from Aldrich Chemical Co. Culture medium for growth of macrophages consisted of Dulbecco's modified Eagle's medium supplemented with 4 mML-glutamine and 10% HICS. Dulbecco's modified Eagle's medium supplemented with 4 mML-glutamine, 10% HICS, vitamins, and essential amino acids was the medium used for macrophage attachment to the microcarrier beads. Activated macrophage studies were performed in medium consisting of Auto-POW-modified Eagle's medium without phenol red, L-glutamine, or NaHCO(3), and 10% HICS, 4 mML-glutamine, vitamins, essential amino acids, 20 mM HEPES, 1 mM sodium pyruvate, 22 mMD-glucose, and 30 mM NaHCO(3).

Microcarrier Bead Preparation

Cytodex 3 (Pharmacia) microcarrier beads (400 mg) were hydrated with 40 ml of 0.01 M phosphate buffer, pH 7.4 (Ca- and Mg-free) for at least 3 h. The average hydrated diameter of the beads was 175 µm, with 50% water content. The beads were washed in 20 ml of the phosphate buffer and then resuspended in approximately 20 ml, after which they were autoclaved for 15 min (250 °C, 15 p.s.i.) and stored at 4 °C. Prior to most experiments, the bead solution was replaced with Dulbecco's modified Eagle's medium supplemented with 4 mML-glutamine, 10% HICS, vitamins, and essential amino acids and left overnight at 37 °C in 5% CO(2).

Macrophage Growth and Attachment

Macrophages from the immortalized cell line RAW264.7 (American Type Tissue Culture Collection) were grown in 100-mm tissue culture dishes and kept in 5% CO(2) at 37 °C. Approximately 2.4 times 10^8 viable macrophages, determined via Trypan blue exclusion, were removed from the culture dishes and suspended in 100 ml of culture medium in a siliconized 100-ml spinner flask (Kontes, Vineland, NJ). The glass was siliconized by adding a 15% solution of dimethyldichlorosilane in toluene, letting the flask sit for 30 min, then rinsing successively with toluene, methanol, and water, and finally baking at 100 °C for 30 min. Microcarrier beads (400 mg) were then added for cell attachment. The beads and cells were stirred at 30 rpm for at least 6 h at 37 °C to allow sufficient time for attachment.

Macrophage Experiments

Following cell attachment to the beads (approximately 50-75% of cells attached), the beads were allowed to settle, and the medium was removed. The beads with the cells were suspended in 100 ml of medium in the reactor described below. To activate the macrophages, LPS (1 µg/ml) and -interferon (200 units/ml) were added. In some experiments, either Mor (1 mM) or SOD (3.2 µM) was also added. The reactor was then placed in an oven at 37 ± 1 °C with stirring at 55 rpm and sterilized gas (5% CO(2), 21.7% O(2), balance N(2)) continuously flowing at 300 standard cm^3/min through the gas phase. The NO concentration was continuously measured, and 0.7-ml samples were withdrawn periodically for NO(2), NO(3), and NMor measurements. Viable cell counts were measured at the beginning of the experiments, near the onset of NO production (approximately 8 h after stimulation), and at the end of the experiments. Viability was determined by taking 2-3 samples (0.7 ml), centrifuging the solution, removing the supernatant, and adding 0.5 ml of trypsin-EDTA (0.25%) to the remaining beads and cells. After 10 min of incubation at 37 °C, 0.5 ml of culture medium was added, and viable cells were determined by Trypan blue exclusion. The beads did not interfere with cell counting in the hemacytometer.

NO Measurements

Fig. 1illustrates the reactor, used as both a spinner flask for the suspension of the beads with macrophages and an NO measuring device. The reactor was an ultrafiltration cell modified as described previously(11) . The aqueous NO concentration was continuously measured via NO permeation through the base of the ultrafiltration cell and into a chemiluminescence detector (Thermedics Detection Inc., Woburn, MA, model TEA-502). The volumetric mass transfer coefficient (k(L)a/V), describing the transport of NO across both the gas-liquid interface and into the detector by physical processes, was 0.00075 s(11) . This coefficient allowed calculation of the rate of physical removal of NO from the aqueous solution, with the removal across the gas-liquid interface accounting for >95% of the physical losses and the remainder entering the detector.


Figure 1: Schematic of reactor used for macrophage studies. A modified 200-ml ultrafiltration cell was used for the suspension culture. Nitric oxide was measured by continuous entry into a chemiluminescence detector via a composite membrane composed of a polydimethylsiloxane (Silastic) membrane laminated to a polytetrafluorethylene (Teflon) sheet; four symmetrically positioned holes in the latter restricted the flow of NO into the detector. Sterilized gas (5% CO(2), 21% O(2), balance N(2)) flowed continuously through the head space (adapted from (11) .).



NO, NO, and NMor Measurements

The concentrations of NO(2) and NO(3) were measured using an automated Griess procedure(12) . NMor was analyzed by extracting it from the sample with an equal volume of dichloromethane, after which the sample was centrifuged for a few minutes to separate the layers. The dichloromethane containing the NMor was analyzed by mass spectrometry carried out on a Hewlett Packard 5989A GC-MS instrument. NMor was chromatographed on fused silica capillary columns coated with HP-1, HP-5 (Hewlett Packard, Palo Alto, CA), or Supelcowax 10 (Supelco, Bellefonte, PA). Helium was the carrier gas with head pressures of about 5-15 p.s.i., depending on the column length and diameter. The mass spectrometer was operated in the selected ion mode at m/z = 116, 86, and 128. NMor was quantitated by normalizing the appropriate peak areas for NMor to that of an internal standard of naphthalene in each injection.


RESULTS

NO Concentrations

Accumulation of NO became evident at approximately 2-5 h following stimulation of the macrophages with LPS and -interferon, which is consistent with previous observations for the onset of NO production(8) . In most experiments steady state NO concentrations were observed after approximately 9 h. The overall behavior of the measured NO concentration was similar for all experiments, whether or not Mor or SOD was present. In a control experiment with unstimulated macrophages, NO accumulation was not observed. Fig. 2shows the NO concentration as a function of time (t) in a typical experiment, with t = 0 indicating the time at which the cells were stimulated.


Figure 2: Concentrations of nitric oxide, nitrite, nitrate, and NMor as a function of time following macrophage stimulation. The data are for experiment number 1M.



NO and NO Formation

Measurable rates of NO(2) and NO(3) formation coincided with the onset of NO accumulation, as shown for a typical experiment in Fig. 2. This temporal correspondence supports the view that extracellular NO leads to extracellular formation of NO(2) and NO(3) and is consistent with previous findings that both NO(2) and NO(3) are formed in the presence of NO-producing cells(4, 5, 6, 7) . In the control experiment using unstimulated macrophages, NO(2) and NO(3) formation was not observed. The slopes of the NO(2) and NO(3) concentration data, corresponding to the formation rates, were essentially constant from a few hours after macrophage stimulation up to and including the period when the NO concentration reached a steady state.

Table 1summarizes the steady state NO concentrations, the formation rates of NO(2) and NO(3), and the rates of physical loss of NO for each of the 10 individual experiments using stimulated macrophages. The suffixes M and S identify experiments in which morpholine or SOD, respectively, was added to the culture medium. The steady state NO concentrations were calculated as averages over the last 6 h, whereas the formation rates of NO(2) and NO(3) were obtained by linear regression of the NO(2) and NO(3) concentration data over approximately the last 8-9 h. (Inclusion of NO(2) and NO(3) data a few hours before the attainment of an NO steady state did not significantly affect the calculated formation rates.) The rates of physical removal of NO from the reactor were calculated using the steady state NO concentrations. Also shown in Table 1are the viable cell concentrations during the NO steady state period, which remained essentially constant. On average, 90% of the cells counted were viable, the number exceeding 80% in all cases. All of the rates shown in Table 1have been normalized by dividing the absolute rate by the number of viable cells.



The effect of SOD on the relative rates of NO(2) and NO(3) formation is shown in Fig. 3. In the absence of SOD, the ratio of NO(3) to NO(2) formation was about 0.9, whether or not morpholine was present; the average for the seven non-SOD experiments was 0.88 ± 0.07. With the addition of SOD, this ratio declined to 0.39 ± 0.09. Using an unpaired t test, the difference in the nitrate to nitrite ratios was significant at the p < 0.01 level. This effect of SOD confirms that O(2), which is scavenged by SOD, enters the extracellular fluid. It is consistent with the previous observation that extracellular SOD prolonged the lifetime of NO in the extracellular surroundings(13) , which is explained by the fact that NO reacts rapidly with O(2)(14) .


Figure 3: Rate of nitrate formation (R) relative to rate of nitrite formation (R). The relative rates were calculated from the ratio of the concentration changes over the last 8-9 h, when the concentrations varied linearly with time. Mean values ± S.E. are shown for the experiments without morpholine or superoxide dismutase (n = 4), with morpholine only (+ Mor; n = 3), or with superoxide dismutase only (+ SOD; n = 3).



NMor Formation

Only the uncharged form of morpholine is available for N-nitrosation to form NMor(15) . Denoting total morpholine as Mor and the uncharged form as Mor^o, the respective concentrations are related by

On-line formulae not verified for accuracy

where the pK at 37 °C is 8.23(16) . For this study [Mor] = 1000 µM, and this remained essentially constant throughout all experiments because <0.2% was converted to NMor. However, the pH decreased over time, mostly during the steady state period for NO, from approximately 7.4 to 7.0. Thus, [Mor^o] did not remain constant but had a range of 60 to 130 µM with an average of 100 µM.

Formation of NMor was observed with stimulated but not with unstimulated macrophages. As exemplified by the data in Fig. 2, the onset of NMor formation coincided with the appearance of NO in the culture medium and with the onset of NO(2) and NO(3) formation. In preliminary experiments we incubated macrophages with 1 mM morpholine for 16-24 h. Morpholine could not be detected in cell extracts, and no change in morpholine concentration was observed in the media, indicating that morpholine entry into the cells was insignificant. Taken together, these findings are consistent with the view that NMor formation is an extracellular process and that NMor is formed via oxidation products of NO, most likely N(2)O(3)(9, 10, 17) .

NO Release Rate

The rate at which NO was released into the culture medium by the stimulated macrophages was calculated from the measured rates of formation of nitrogen-containing species in the solution, with a correction for physical losses of NO from the reactor. A nitrogen balance on the system shows that the rate of NO release into the solution (S, expressed as mol/unit of volume/unit of time) is given by

On-line formulae not verified for accuracy

The first three terms on the right hand side of represent the accumulation of aqueous NO and its major end products, whereas the last term represents the removal of NO by physical processes. The various NO(x) intermediates were not included because their concentrations are much smaller than the species in . For these studies, NMor formation was negligible compared with NO(2) and NO(3) formation, so that NMor was also excluded from . The products of other possible nitrosation reactions, such as with amines (18) or thiols(17, 19) , were assumed to be negligible as well.

Once a steady state NO concentration was obtained (at 9 h), it was observed that all of the terms in were essentially constant. Thus, a constant NO concentration in the extracellular fluid corresponded with a constant NO release rate. This steady release rate was calculated by adding the rates of NO(2) and NO(3) formation and the rate of physical loss of NO, as given in Table 1. The rate of NO release averaged 6.0 ± 0.4 pmol s (10^6 cells), which is comparable with results from previous macrophage studies(5, 7) . An average of 13% of the NO released by the macrophages was removed by physical processes, primarily by diffusion across the gas-liquid interface (into the head space).


DISCUSSION

It is clear that activated macrophages produce NO, which can react extracellularly to form NO(2), NO(3), and NMor. The question that we address now is whether or not the rate constants of pertinent reactions measured in cell-free systems can account for the rates at which these products are formed in the presence of NO-generating cells.

Basic Reaction Scheme

The formation of NO(2) and NMor in aqueous solutions at physiological pH in the presence of NO and O(2) has been modeled successfully using the following set of reactions (9) .

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Reactions 3-5 represent the oxidation of NO by molecular oxygen; at physiological pH, where the equilibrium in reaction 5 is shifted far to the right, the main product is NO(2). In reaction 6, RH represents any nitrosatable species, including various amines and thiols. For the present experiments we assume that RH is predominantly Mor^o and that RNO is NMor. Reaction 7 is essentially an enhanced hydrolysis resulting from the reaction of N(2)O(3) with various anions, represented as X. It has been shown recently that both chloride and phosphate participate in such a reaction at physiological pH, and rate constants for these reactions have been measured(9) . We will refer to reactions 3-7 as the ``basic reaction scheme.'' As will be shown, an explanation of the present results requires that the basic scheme be supplemented by several reactions involving superoxide (O(2)). In particular, it is noteworthy that NO(3) formation via the reaction of NO and O(2) has not been observed(11, 20) , so that the basic scheme contains no pathway for formation of NO(3).

Nitrite Formation from Basic Scheme

Before considering the effects of superoxide, we examine what the basic reaction scheme implies concerning the rate of nitrite formation. The rate of NO(2) formation at physiological pH has been shown to be given by (9) .

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The effects of Mor, chloride, and phosphate are shown explicitly, whereas the summation terms involving RH and X represent other possible nitrosation and anion reactions of the types represented by reactions 6 and 7, respectively.

To apply to the present data, it is necessary to have values for A and [O(2)]. A precise estimate for A is difficult to obtain because the cell culture medium contained numerous anions and nitrosatable species. However, implies that no matter which species are present, 1 leq A leq 2. At one extreme, if the nitrosation reactions (involving the k(6) terms) are dominant, then A = 1. At the other extreme, if the terms corresponding to regular and/or enhanced hydrolysis of N(2)O(3) are dominant (the k(4) and k terms, respectively), then A = 2. If neither extreme applies, then A will have an intermediate value. Assuming that the summation terms in were negligible and utilizing previously reported rate constants at 37 °C (9) together with the average value of [Mor^o] (100 µM) and the culture medium concentrations of chloride (0.11 M) and phosphate (0.9 mM), the calculated values of A were 2.0 and 1.8 for the experiments without or with morpholine, respectively. Thus, even when Mor was present, we estimate that the hydrolysis of N(2)O(3) had a more important effect on the rate of NO(2) formation than did the nitrosation reactions. It was assumed for the calculations with that [O(2)] = 200 µM, which is 10% lower than the O(2) concentration at saturation (220 µM at 37 °C).

Using the measured NO concentrations it was found that, on average, accounted for only 47 ± 6% of the measured NO(2) formation. Although there was a certain amount of variability, in none of the experiments did the predicted rate exceed or even closely approach that measured. If lower values of A had been assumed, the discrepancies would have been even larger. To check the assumed oxygen level, [O(2)] was estimated from a steady state balance for O(2), where the transport rate of O(2) into the solution was equated to the sum of the basal O(2) consumption rate and the O(2) consumed for the production of NO and O(2). (The rate of O(2) production was inferred as described below.) Based on published O(2) consumption rates for activated alveolar macrophages(7) , it was estimated that [O(2)] could be as low as 180 µM, or 10% lower than the value used in the calculations. From , a ± 10% uncertainty in [O(2)] leads to a similar uncertainty in the predicted formation rate of NO(2). This level of uncertainty does not affect the conclusions, so that further quantitation of the O(2) concentration was deemed unnecessary. Because the cell volume was only 0.4% of the total aqueous volume in the reactor, NO(2) formation from the intracellular reaction of NO with O(2) should have been negligible. We conclude that the basic reaction scheme accounts for only half of the NO(2) formed and that some other pathway for NO(2) formation must have been present.

Nitrite Formation from Superoxide

It was recently reported (26) (^2)that ONOO, which is a product of the reaction of NO and O(2), reacts with NO. A reaction between NO and peroxynitrite is suggested also by the ability of NO to partially inhibit peroxynitrite-mediated benzoate hydroxylation(21) . One of the products of the reaction of NO with ONOO has been identified as NO(2), which suggests that this reaction may account for the unexplained NO(2) formation in the present study. According to the stoichiometry, the other product is NO(2). Adopting this hypothesis, we add to the basic scheme the reactions

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Adding reactions 10 and 11 to reactions 4 and 5, it is found that the overall stoichiometry for NO(2) formation by this pathway is

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Thus, only 1 mol of O(2) is consumed in forming three moles of NO(2). With regard to the kinetics, the rate constant for peroxynitrite formation at 37 °C is k = 6.7 times 10^9M s(14) , but there is no published value for k. Thus, reactions 10 and 11 provide a plausible explanation for the excess NO(2) formation, but a value for k is needed to provide a quantitative test of this hypothesis.

Nitrate Formation

The additional reactions needed to account for NO(3) formation and to describe the scavenging of O(2) by SOD are

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The rate constants are k = 1 s(22) and k(14) = 2.5 times 10^9M s(23) . We should note that the actual species represented as ONOO in reactions 10, 11, and 13 may include its acidic form, peroxynitrous acid, and/or isomeric forms (24) , and thus the rate constants stated here are composites of more elementary rate and equilibrium constants and are likely to vary with pH. The constant k(14) is defined in terms of the bimolecular reaction of O(2) with SOD; that is, the rate of O(2) removal per unit volume is given by k(14)[O(2)][SOD].

The measured effect of SOD on the rate of NO(3) formation provides a test of the hypothesis that NO(3) formation was predominantly extracellular. Presumably, SOD added to the medium would not affect intracellular formation of NO(3), so that any inhibitory effect of SOD is attributable to extracellular reactions. Let R be the rate of nitrate formation per unit of reactor volume (e.g. in nM/s). Averaging the respective results of the SOD and non-SOD experiments in Table 1, it is found that (R)/(R) = 0.39. That is, the addition of 3.2 µM SOD to the medium reduced the rate of NO(3) formation to about 40% of its value in the absence of added SOD. The theoretical effect of adding this amount of SOD is estimated as follows. The rate of NO(3) formation is proportional to [ONOO] (from reaction 13), which in turn is approximately proportional to [NO][O(2)] (reaction 10). Assuming that [O(2)] is directly proportional to the concentration of viable cells and inversely proportional to the rate at which O(2) is consumed by reactions 10 and 14, it follows that

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where n and n are the cell counts (cell concentrations) with and without added SOD, respectively. Implicit in is the assumption that SOD added to the medium does not affect the amount of O(2) released per cell. Using average values from Table 1, gives (R)/(R) = 0.27, in reasonable agreement with the experimental value. Thus, assuming that all of the NO(3) formation was extracellular, the observed degree of inhibition of NO(3) formation by SOD was approximately that expected from the published rate constants for reactions 10 and 14. A more precise calculation would require a rate constant for reaction 11.

Superoxide Release by Macrophages

An overall mass balance for O(2) provides an estimate of the rate at which the cells released O(2) into the medium. In the absence of SOD and with sufficient NO present, any O(2) released into the medium should be consumed in forming NO(3) (reactions 10 and 13) or ``excess'' NO(2) (reactions 10 and 11). Thus, the mass balance for O(2) is

On-line formulae not verified for accuracy

where f is the fraction of the total NO(2) production due to the superoxide/peroxynitrite pathway. The factor comes from the stoichiometry in reaction 12. For the seven experiments without SOD, f = 0.59 ± 0.07. Using the average values for the non-SOD experiments in Table 1, gives S/n = 2.9 pmol s (10^6 cells). The average rate of NO release into the medium in these same experiments was S/n = 5.8 pmol s (10^6 cells), so that S/S = 0.51. In other words, the rate of O(2) release into the medium is inferred to be half that of NO.

The rate of O(2) formation has been measured recently using the same cell line under similar conditions except that the cells were plated in tissue culture wells(25) . The amounts of O(2), NO(3), and NO(2) formed in various 2-h intervals were determined by various assays. It was found that LPS and -interferon did not elicit an oxidative burst and that O(2) formation was constant over the entire experimental period. For the time interval most comparable with the present study (6-8 h after stimulation), the ratio of O(2) to NO release (as assessed from the sum of NO(3) and NO(2) formation) was 0.48. This is in excellent agreement with the release ratio calculated here using .

Relatively little of the O(2) (about 18%) was calculated to result in NO(2) formation. With a O(2)/NO release ratio of 0.5, and with almost all of the O(2) participating in NO(3) formation, roughly half of the NO was converted to NO(3) when SOD was not present. Thus, in the absence of SOD, the rates of NO(3) and NO(2) formation were roughly comparable ( Table 1and Fig. 3). In general, the NO(3)/NO(2) concentration ratio in the medium should be very sensitive to the O(2)/NO release ratio obtained with a given cell culture. Given that macrophages in culture can be stimulated to form varying amounts of NO, while presumably releasing nearly constant amounts of O(2), it is understandable that there would be variations in [NO(3)]/[NO(2)] from study to study. Reported values of [NO(3)]/[NO(2)] for macrophage cultures range from 0.67 to 1.0(5, 8) . Of course, in vivo or with other systems in vitro, other reactions will influence the ultimate disposition of NO. For example, in the presence of oxyhemoglobin or oxymyoglobin, NO and NO(2) are oxidized completely to NO(3)(20) .

One of the assumptions underlying was that, when SOD was not present in the culture medium, extracellular O(2) reacted only with NO. For a steady state to exist, O(2) must be consumed by extracellular reactions at the same rate at which it is released by the cells into the culture medium. The maximum rate at which O(2) can react with NO is equivalent to the NO release rate. Therefore, if O(2) is able to react only with NO, there will be a continual increase in the O(2) concentration whenever the O(2) release rate is greater than the NO release rate. In reality, other reactions would eventually become significant, such as the uncatalyzed dismutation of O(2)(7) .

Predicted versus Observed Rates of NMor Formation

The rate of nitrosomorpholine formation was predicted using

On-line formulae not verified for accuracy

An expression for [N(2)O(3)] was derived by applying pseudo-steady state approximations to N(2)O(3) and NO(2), both of which are trace intermediates. The result is

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In computing [N(2)O(3)] there was little error in using the average value of [Mor^o] of 100 µM, because the term in involving [Mor^o] was much smaller than that involving [Cl]. However, this same approximation could not be applied to the [Mor^o] term in . Instead, [Mor^o] was related to [Mor] (a constant) and pH using , and the data for each morpholine experiment were used to express pH as a linear function of time. was integrated numerically between the onset of the steady state NO period and the end of the experiment to predict the changes in NMor concentration.

Predicted changes in NMor concentration are compared with the measured values in Fig. 4, the results being expressed as the ratio of the predicted to the measured value for the three individual experiments. The results obtained using the full kinetic model are shown by the solid bars. On average, the predicted values were about 6 times too large, implying that the actual value of [N(2)O(3)] was about of that suggested by the kinetic model. Both chloride and phosphate participate in the enhanced hydrolysis represented by reaction 7, which has the effect of reducing [N(2)O(3)] well below its theoretical value in the absence of these anions(9) . If these recently discovered effects of chloride and phosphate had been neglected, the overpredictions would have been approximately 25-fold, as shown by the open bars in Fig. 4.


Figure 4: Ratio of predicted to measured rates of NMor formation in individual experiments. Predicted values are shown based on the full kinetic model (solid bars) and when the inhibitory effects of chloride and (phosphate (P(i)) are excluded (open bars).



Concentrations of NMor were predicted also using data from the macrophage study of Miwa et al.(4) . Inputs for the calculation included the reported pH of 7.4, [Mor] = 5 mM, and [Cl] = 0.11 M (based on the culture medium used). The NMor concentration predicted for the conditions of Miwa et al. was a factor of 14 larger than the measured value, in approximate agreement with the results in Fig. 4. If the pH in the experiments of Miwa et al. eventually decreased below 7.4, as it did here, the overprediction factor would be smaller than 14, and the agreement with Fig. 4would be even better. It should be noted that the experiments of Miwa et al. were over a period of 72 h, as compared with 14-19 h in the present study. Also, the concentrations of LPS and -interferon used by Miwa et al. were higher than in the present experiments, which may have led to a somewhat different ratio of NO(3) to NO(2) formation rates. Although the experimental conditions were not exactly the same, it is clear that the model consistently overpredicts NMor formation in both cases. It appears that in macrophage cultures there are other reactions that compete for N(2)O(3), thereby lowering the concentration of this trace species.

Conclusions

It was found that, when applied to the extracellular fluid in a macrophage culture, the basic reaction scheme used previously to describe NO kinetics in oxygenated buffer solutions (reactions 3-7) was incomplete in at least two respects. In particular, a kinetic analysis showed that only half of the NO(2) formed could be accounted for by the reaction of NO with molecular oxygen. Moreover, the basic scheme contains no pathway for the formation of NO(3). The present results are explained by including certain reactions due to superoxide (reactions 10, 11, 13, and 14). The most novel aspect of the proposed scheme is reaction 11, in which the reaction of NO with ONOO provides an additional source for NO(2). Independent measurements of the rate constant for this reaction are needed to verify that this is the source of the excess NO(2). The kinetic model overpredicted the rate of nitrosomorpholine formation by a factor of 6, suggesting that competition from reactions not included in the present scheme lowered the concentration of N(2)O(3) by this factor. Further work is needed to determine which of the many possible reactions involving N(2)O(3) are most significant in this regard.


FOOTNOTES

*
This work was supported by Grant PO1-CA26731 from the National Cancer Institute and Grant ES 02109 from the National Institute of Environmental Health Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: School of Chemical Engineering, Oklahoma State University, Stillwater, OK 74078.

To whom correspondence should be addressed: Dept. of Chemical Engineering, Room 66-509, Massachusetts Inst. of Technology, Cambridge, MA 02139. Tel.: 617-253-4535; Fax: 617-258-8224; wmdeen@mit.edu.

(^1)
The abbreviations used are: NMor, N-nitrosomorpholine; SOD, superoxide dismutase; HICS, heat-inactivated calf serum; LPS, lipopolysaccharide; Mor, morpholine.

(^2)
One of the reaction products was identified as NO(2) subsequent to publication of (26) (J. P. Crow and J. S. Beckman, personal communication).


ACKNOWLEDGEMENTS

We appreciate the assistance of Dr. John S. Wishnok with the measurements of N-nitrosomorpholine concentration.


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