(Received for publication, February 2, 1995; and in revised form, September 15, 1995)
From the
To investigate the fate of nitric oxide (NO) synthesized by
activated macrophages, the concentrations of NO and its principal
reaction products, nitrite (NO) and
nitrate (NO
), 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
and
NO
, 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
formed could be accounted for by
the reaction of NO with O
. It is known that NO reacts with
superoxide (O
) to give peroxynitrite
and that NO also reacts with peroxynitrite to yield
NO
, so that the latter reaction may
explain the ``excess'' NO
formation. Adding superoxide dismutase to the medium markedly reduced
the ratio of NO
to
NO
, consistent with the hypothesis that
NO
in the medium results primarily from
the extracellular reaction of NO with
O
. 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
.
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
(NO
), 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
O
with various
secondary amines(2) . Cytotoxic and mutagenic effects may
result also from reactions involving NO and superoxide
(O
), such as lipid peroxidation via
peroxynitrite (ONOO
), an intermediate in the reaction
of NO with O
(3) .
Although
there has been much interest in the biological effects of NO and
species formed by the reactions of NO with O and
O
, 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
, resulting in the formation of
NO
and NO
as end products in various ratios (4, 5, 6, 7, 8) .
Intermediates such as N
O
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), nitrate
(NO
), and N-nitrosomorpholine
(NMor) (
)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
, NO
, 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
O
(9, 10) . Accordingly, the
rate of formation of NMor was used to indirectly assess the
concentration of N
O
. Superoxide dismutase
(SOD), which scavenges O
, was added to
some of the macrophage cultures. In oxygenated buffer solutions
NO
is not formed from NO in the absence
of O
(11) , and this is true
also for cell culture medium. Thus, the effect of SOD on the relative
rates of NO
and
NO
formation provides an indication of
the levels of O
present in the
extracellular solution.
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
, 21% O
, balance
N
) flowed continuously through the head space (adapted from (11) .).
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.
Table 1summarizes the steady state NO concentrations,
the formation rates of NO and
NO
, 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
and
NO
were obtained by linear regression of
the NO
and NO
concentration data over approximately the last 8-9 h.
(Inclusion of NO
and
NO
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 and
NO
formation is shown in Fig. 3.
In the absence of SOD, the ratio of NO
to
NO
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
, 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
(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).
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] 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 and NO
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
O
(9, 10, 17) .
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 intermediates were not
included because their concentrations are much smaller than the species
in . For these studies, NMor formation was negligible
compared with NO
and
NO
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
and NO
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
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).
It is clear that activated macrophages produce NO, which can
react extracellularly to form NO,
NO
, 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.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
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. In reaction 6, RH represents
any nitrosatable species, including various amines and thiols. For the
present experiments we assume that RH is predominantly Mor
and that RNO is NMor. Reaction 7 is essentially an
enhanced hydrolysis resulting from the reaction of N
O
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
). In particular, it is noteworthy
that NO
formation via the reaction of NO
and O
has not been observed(11, 20) , so
that the basic scheme contains no pathway for formation of
NO
.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
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].
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
A
2. At one extreme, if the nitrosation reactions
(involving the k
terms) are dominant, then A = 1. At the other extreme, if the terms corresponding to
regular and/or enhanced hydrolysis of N
O
are
dominant (the k
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
] (
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
O
had a more
important effect on the rate of NO
formation than did the nitrosation reactions. It was assumed for
the calculations with that [O
]
= 200 µM, which is 10% lower than the O
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 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
] was estimated from a steady state balance for
O
, where the transport rate of O
into the
solution was equated to the sum of the basal O
consumption
rate and the O
consumed for the production of NO and
O
. (The rate of
O
production was inferred as described
below.) Based on published O
consumption rates for
activated alveolar macrophages(7) , it was estimated that
[O
] could be as low as 180 µM, or
10% lower than the value used in the calculations. From , a
± 10% uncertainty in [O
] leads to a
similar uncertainty in the predicted formation rate of
NO
. This level of uncertainty does not
affect the conclusions, so that further quantitation of the O
concentration was deemed unnecessary. Because the cell volume was
only
0.4% of the total aqueous volume in the reactor,
NO
formation from the intracellular
reaction of NO with O
should have been negligible. We
conclude that the basic reaction scheme accounts for only half of the
NO
formed and that some other pathway for
NO
formation must have been present.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Adding reactions 10 and 11 to reactions 4 and 5, it is found
that the overall stoichiometry for NO formation by this pathway is
On-line formulae not verified for accuracy
Thus, only 1 mol of O is
consumed in forming three moles of NO
.
With regard to the kinetics, the rate constant for peroxynitrite
formation at 37 °C is k
= 6.7
10
M
s
(14) , but there is no published value for k
. Thus, reactions 10 and 11 provide a plausible
explanation for the excess NO
formation,
but a value for k
is needed to provide a
quantitative test of this hypothesis.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
The rate constants are k = 1
s
(22) and k
=
2.5
10
M
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
is defined
in terms of the bimolecular reaction of O
with SOD; that is, the rate of O
removal per unit volume is given by k
[O
][SOD].
The measured effect of SOD on the rate of NO formation provides a test of the hypothesis that
NO
formation was predominantly
extracellular. Presumably, SOD added to the medium would not affect
intracellular formation of NO
, 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
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
formation is proportional to
[ONOO
] (from reaction 13), which in turn is
approximately proportional to
[NO][O
] (reaction
10). Assuming that [O
] is
directly proportional to the concentration of viable cells and
inversely proportional to the rate at which O
is consumed by reactions 10 and 14, it follows that
On-line formulae not verified for accuracy
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
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
formation was extracellular, the observed degree of inhibition of
NO
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.
On-line formulae not verified for accuracy
where f is the fraction of the total
NO 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
cells)
. The average rate of NO release into the
medium in these same experiments was S
/n = 5.8 pmol s
(10
cells)
, so that S
/S
= 0.51. In other words, the rate of O
release into the medium is inferred to be half that of NO.
The
rate of O 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
, NO
,
and NO
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
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
to NO release (as assessed from the
sum of NO
and NO
formation) was 0.48. This is in excellent agreement with the
release ratio calculated here using .
Relatively little
of the O (about 18%) was calculated to
result in NO
formation. With a
O
/NO release ratio of 0.5, and with
almost all of the O
participating in
NO
formation, roughly half of the NO was
converted to NO
when SOD was not present.
Thus, in the absence of SOD, the rates of NO
and NO
formation were roughly
comparable ( Table 1and Fig. 3). In general, the
NO
/NO
concentration ratio in the medium should be very sensitive to the
O
/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
, it is
understandable that there would be variations in
[NO
]/[NO
]
from study to study. Reported values of
[NO
]/[NO
]
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
are oxidized completely to
NO
(20) .
One of the
assumptions underlying was that, when SOD was not present
in the culture medium, extracellular O reacted only with NO. For a steady state to exist,
O
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
can react with NO is equivalent to
the NO release rate. Therefore, if O
is
able to react only with NO, there will be a continual increase in the
O
concentration whenever the
O
release rate is greater than the NO
release rate. In reality, other reactions would eventually become
significant, such as the uncatalyzed dismutation of
O
(7) .
On-line formulae not verified for accuracy
An expression for [NO
] was
derived by applying pseudo-steady state approximations to
N
O
and NO
, both of which are trace
intermediates. The result is
On-line formulae not verified for accuracy
In computing [NO
] there was
little error in using the average value of [Mor
]
of 100 µM, because the term in involving
[Mor
] was much smaller than that involving
[Cl
]. However, this same approximation
could not be applied to the [Mor
] term in . Instead, [Mor
] 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
[NO
] 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
O
] 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) 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
to
NO
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
O
, thereby lowering the
concentration of this trace species.