(Received for publication, April 11, 1994; and in revised form, November 10, 1994)
From the
A Clark-type nitric oxide-sensitive electrode was used for
electrochemical determination of NO oxidation kinetics. Reaction with
molecular oxygen followed second-order rate law with respect to NO with
an overall rate constant of 9.2 ± 0.33 10
M
s
.
Tetrahydrobiopterin, an essential cofactor of NO synthases, was found
to induce rapid oxidation of NO in a 1:1 stoichiometry. The reaction
required the presence of oxygen, was zero order with respect to NO and
first order with respect to tetrahydrobiopterin, completely blocked by
5,000 units/ml superoxide dismutase, and mimicked by a
superoxide-generating system. Purified brain NO synthase produced no
detectable NO unless high amounts of superoxide dismutase were present.
NO synthase-catalyzed citrulline formation was inhibited by superoxide
dismutase (5,000 units/ml) in an oxyhemoglobin-sensitive manner,
indicating that NO induces feedback inhibition of NO synthase.
NO-stimulated soluble guanylyl cyclase was inhibited by
tetrahydrobiopterin at half-maximally active concentrations of 2
µM. The present data suggest that NO is inactivated to
peroxynitrite by superoxide generated in the course of
tetrahydrobiopterin autoxidation.
The biologic effects of NO are almost exclusively determined by its chemical properties. As a free radical, NO reacts rapidly with molecular oxygen, superoxide, and iron-containing proteins. Reaction with oxygen results in the formation of as yet poorly defined reactive intermediates causing oxidative damage of DNA(1, 2) . Reaction with superoxide is extremely fast and generates peroxynitrite, which is rapidly inactivated when protonated to the corresponding peroxynitrous acid(3) . Decomposition of peroxynitrous acid involves formation of cytotoxic intermediates with chemical and biological properties similar to hydroxyl radical(4) . Finally, the activity of a wide variety of iron-containing enzymes is affected by NO. Several non-heme iron proteins, such as cis-aconitase or ribonucleotide reductase are inhibited(5) , whereas heme-containing soluble guanylyl cyclase is stimulated several hundredfold upon binding of NO to its regulatory prosthetic heme group(6) . Microsomal cytochromes P-450, however, which contain a catalytically active heme, are completely inhibited at submicromolar concentrations of NO(7) .
The enzymes involved in
biosynthesis of NO from L-arginine, the NO synthases (NOS), ()have been identified as cytochrome P-450-like
hemeproteins(8, 9, 10, 11) , and it
is conceivable, therefore, that NO blocks its own formation. We have
recently studied the possible feedback inhibition of purified brain
NOS, but observed no appreciable inhibition of citrulline formation
upon addition of high concentrations of authentic NO or drugs believed
to release NO. Moreover, reaction rates were not enhanced in the
presence of oxyhemoglobin, an effective scavenger of NO(12) .
However, several reports from different groups demonstrate inhibition
of NO synthesis by NO in brain cytosol, endothelial cells, and
macrophages (13, 14, 15, 16, 17) .
Albeit these studies used fairly high concentrations of NO to produce
inhibition, the results clearly conflict with our data.
A major
drawback of previous work on feedback inhibition of NOS was that actual
NO concentrations have not been measured in the incubation mixtures. In
the present study we have used an NO-sensitive Clark-type electrode for
NO measurements under various conditions and found that the NOS
cofactor Hbiopterin induced a rapid oxidation of NO. This
pronounced effect was apparently due to generation of superoxide in the
course of H
biopterin autoxidation. Interestingly, purified
NOS produced no detectable amounts of NO, unless high concentrations of
SOD had been added, regardless whether H
biopterin was
present or not.
The donor compound
DEA/NO (21) was used to generate defined amounts of NO for
determination of NO:Hbiopterin stoichiometry. Following
application of DEA/NO (20-120 nmol), NO concentrations rapidly
increased to 3-9 µM, followed by a slow decrease of
the signals. The areas under the curve were calculated by integration
from t
= 0 (addition of DEA/NO) and t
(defined as the time point at which NO
concentrations had decreased to 0.5 µM (
30 min)).
There was a linear relationship between this area and NO generated from
20 to 120 nmol of DEA/NO. Consumption of NO was measured in the
presence of 40-120 nmol of DEA/NO and 10-40 nmol of
H
biopterin. NO:oxygen stoichiometry was measured by
electrochemical determination of oxygen consumption in the presence of
0.1-0.2 mM DEA/NO. Initial oxygen concentrations were
210 ± 5.5 µM (n = 20).
We obtained linear responses by calibration of a Clark-type NO-sensitive electrode with NO generated from 0.05 up to 10 µM acidified nitrite. As an example, Fig. 1shows representative traces obtained by calibration of the electrode with 0.4-2 µM nitrite. A sensitivity of 3.85 nM/pA was calculated from the slope of the linear curve fit shown in the inset to Fig. 1. This is in good accordance with a previously reported sensitivity of 3.92 ± 0.23 nM/pA(27) . Since fluctuations of basal currents were usually 2-3 pA, the detection limit of our setup was approximately 20 nM NO. For unknown reasons, basal currents were quite unstable occasionally, but grounding of the operator with a stainless steel wire markedly improved stability in these cases. It is possible, therefore, that taking additional precautions against interferences by surrounding electromagnetical fields could increase sensitivity. The method appears to be specific for NO, since no response of the electrode was observed in the presence of high concentrations of nitrite, nitrate, peroxynitrite, hydrogen peroxide, or a superoxide-generating system (not shown).
Figure 1:
Calibration of the NO electrode. At the
indicated time points (), 7.2 µl of a 0.1 mM solution of KNO
were added to 1.8 ml of a
helium-deoxygenated solution of 0.14 M K
SO
and 0.1 M KI in 0.1 M H
SO
yielding NO concentrations of 0.4, 0.8, 1.2, 1.6, and 2.0
µM (2 KNO
+ 2 KI + 2
K
SO
+ 2 H
SO
2 NO + I
+ 2 H
O + 4
K
SO
). Inset, correlation between NO
concentration and output current of the electrode (3.85
nM/pA).
Fig. 2shows
representative traces obtained with authentic NO. Addition of 4 µl
of an NO solution (2 mM) to total volumes of 1.8 ml
induced a pronounced response of the electrode corresponding to a
maximum of
1.8 µM NO, followed by a decrease of the
signal according to second order kinetics (see Fig. 2, inset). Measurement of oxygen uptake in the presence of
defined amounts of NO released from the NO donor DEA/NO (21) showed that 1 molecule of oxygen consumed 3.92 ±
0.23 (n = 5) molecules of NO. Assuming a first order
rate law with respect to oxygen(22) , we obtained a third order
rate constant for NO autoxidation of 9.2 ± 0.33
10
M
s
(n = 11), which was not changed by 5,000 units/ml SOD (9.5
± 0.54
10
M
s
; n = 4).
Figure 2:
Autoxidation of NO. Experiments were
performed at ambient temperature in the absence (-) and
presence of 5,000 units/ml SOD (). At
time point zero, 4 µl of a saturated NO solution (
2
mM) were added to 1.8 ml of 50 mM triethanolamine/HCl
buffer, pH 7.0, and changes in NO concentrations were monitored over 13
min (
350 data points). Inset, replot of the original data
(2.5 to 13 min;
230 points) according to second-order rate law.
The original tracing and the plot shown in the inset are representative
of 11 (Control) or four (SOD) similar
experiments.
Rates of NO
oxidation were markedly increased by addition of
Hbiopterin. As can be seen from the representative
experiment shown in Fig. 3, oxidation kinetics was changed from
second to zero order with respect to NO, suggesting that NO itself was
not involved in the rate-limiting reaction step that induced its rapid
oxidation. The reaction was first order with respect to
H
biopterin, and gassing the buffer with helium for 15 min
prior to the experiments completely blocked the effect of
H
biopterin (not shown), indicating that NO oxidation was
triggered by a reaction of molecular oxygen with
H
biopterin. Experiments in which DEA/NO was used for
release of defined amounts of NO revealed that 1 mol of
H
biopterin consumed 0.98 ± 0.10 (n =
23) mol of NO. However, the pteridine became substoichiometrically
active in the presence of reducing compounds. Pure NADPH (0.1
mM), for instance, did not affect autoxidation of NO but
markedly decreased H
biopterin:NO stoichiometry to at least
1:10.
Figure 3:
Effect of Hbiopterin on NO
oxidation. Experiments were performed at ambient temperature in the
absence (-) and presence of 5,000 units/ml SOD
(
). At time point zero, 4 µl of a
saturated NO solution (
2 mM) were added to 1.8 ml of 50
mM triethanolamine/HCl buffer, pH 7.0. At the maximum of the
NO peak, a solution of H
biopterin (30 µM final
concentration) was added. Inset, replot of the data (2.5 to 15
min;
270 points) obtained in the presence of SOD according to
second-order rate law. The original tracing and the plot shown in the inset are representative of 11 (Control) or 4 (SOD) similar experiments.
Since SOD (5,000 units/ml) completely prevented
Hbiopterin-induced oxidation of NO (Fig. 3), we
speculated that the effect was mediated by superoxide generated via
H
biopterin autoxidation. Two sets of experiments were
performed to substantiate this hypothesis. First, we determined rates
of H
biopterin autoxidation directly by quantitative HPLC
analysis. Autoxidation was first order with respect to
H
biopterin (k
= 3.03 ±
0.37
10
s
; n = 5). Assuming first order kinetics of oxygen
consumption(23) , we obtained a rate constant for
H
biopterin autoxidation of 1.44 ± 0.18 M
s
(n =
5), which was comparable with the respective constant obtained by
determination of NO oxidation in the presence of H
biopterin
(1.88 ± 0.15 M
s
; n = 11). Second, we tested whether generation of
superoxide by hypoxanthine/xanthine oxidase mimicked
H
biopterin-induced oxidation of NO. As shown in Fig. 4, addition of xanthine oxidase (1.67 milliunits) to an NO
solution containing 1 mM hypoxanthine induced an
SOD-inhibitable switch of NO oxidation from second to zero order
kinetics, which was very similar to that observed in the presence of
H
biopterin. Consumption of NO was 10.3 ± 0.73 nM s
(n = 4) under these
conditions, demonstrating that rates of zero order NO oxidation were a
good measure for superoxide production by the hypoxanthine/xanthine
oxidase system. These data suggest that H
biopterin-induced
generation of superoxide accounts for the rapid oxidation of NO in the
presence of the NOS cofactor H
biopterin.
Figure 4:
Effect of hypoxanthine/xanthine oxidase on
NO oxidation. Experiments were performed at ambient temperature in the
absence (-) and presence of 5,000 units/ml SOD
(). At time point zero, 4 µl of a
saturated NO solution (
2 mM) were added to 1.8 ml of 50
mM triethanolamine/HCl buffer, pH 7.0, containing 1 mM hypoxanthine. After reaching the maximal NO concentration, 1.67
milliunits of xanthine oxidase were added. Inset, replot of
the data (2.5-15 min;
270 points) obtained in the presence
of SOD according to second-order rate law. The original tracing and the
plot shown are representative of four.
Since these
findings appeared to be of potential relevance to the biology of NO, we
investigated whether the biological activity of NO was affected by
Hbiopterin. Fig. 5shows that H
biopterin
produced a concentration-dependent inhibition of purified soluble
guanylyl cyclase, which had been stimulated with 1 µM GSNO. Half-maximal effects were observed at 2 µM H
biopterin, and inhibition was complete at 0.1
mM.
Figure 5:
Effect of Hbiopterin on
NO-induced cGMP formation by purified soluble guanylyl cyclase. Soluble
guanylyl cyclase purified from bovine lung (0.15 µg) was incubated
at 37 °C for 10 min in 50 mM triethanolamine/HCl buffer,
pH 7.5 in the presence of 1 µM GSNO and increasing
concentrations of H
biopterin as described under
``Experimental Procedures.'' Data are means ± S.E. of
three experiments performed in triplicate.
Taking into account the role of Hbiopterin
as essential cofactor of NOS, we measured enzymatically produced NO
with the NO-sensitive electrode. As shown in Fig. 6(upper
panel), we observed no detectable signal by incubation of purified
NOS with L-arginine and requisite cofactors in the absence and
presence (0.1-1 µM) of exogenously added
H
biopterin. However, subsequent addition of 5,000 units/ml
SOD resulted in the generation of NO at initial rates of approximately
20 nM s
, yielding steady state
concentrations of
0.3 µM NO after about 1 min. The lower panel in Fig. 6shows a representative experiment
performed in the presence of SOD. No response of the electrode was
observed in the absence of calmodulin, addition of calmodulin produced
a signal corresponding to
0.1 µM NO, and this was
further increased to
0.3 µM in a
concentration-dependent manner by added H
biopterin. The
half-maximally active concentration of H
biopterin was
approximately 0.1 µM and thus similar to the potency of
the pteridine determined previously with other
methods(24, 28) .
Figure 6:
Electrochemical detection of
enzymatically formed NO. Experiments were performed at ambient
temperature in 0.5 ml of 50 mM triethanolamine/HCl buffer, pH
7.0, containing 0.5 mM CaCl, 1 mML-arginine, 0.3 mM NADPH, 5 µM FAD,
5 µM FMN, and 5 µg of purified brain NOS. Original
tracings of experiments performed in the absence (upper panel)
and presence of 5,000 units/ml SOD (lower panel) are shown.
Where indicated, calmodulin (10 µg/ml), H
biopterin
(0.1-100 µM), or SOD (5,000 units/ml) were added
(final concentrations in parentheses). The tracings shown are
representative of three.
These results demonstrated that NOS produced detectable amounts of NO only when considerably high concentrations of SOD were present. We speculated that this may explain the controversy regarding feedback inhibition of NOS by NO and measured formation of citrulline by the purified enzyme in the absence and presence of 5,000 units/ml SOD. As shown in Fig. 7, the enzyme was inhibited to 51.2% of controls in the presence of SOD, and this effect was completely prevented by 20 µM oxyhemoglobin. SIN-1 (1 mM) had a slight, oxyhemoglobin-insensitive inhibitory effect in the absence of SOD (75.5% of controls), but rates of citrulline formation were reduced down to 26.6% of untreated controls when both SIN-1 and SOD were present. Again, the inhibitory effect of SOD was not observed in the presence of 20 µM oxyhemoglobin.
Figure 7: Effects of oxyhemoglobin on SOD-induced inhibition of NOS. Purified brain NOS (0.2 µg) was incubated at 37 °C for 10 min in 50 mM triethanolamine/HCl buffer, pH 7.5, in the absence and presence of SOD (5,000 units/ml), oxyhemoglobin (20 µM) or SIN-1 (1 mM) as described under ``Experimental Procedures.'' Data are means ± S.E. of five experiments performed in duplicates. OxyHb, oxyhemoglobin.
Electrochemical detection with a Clark-type NO-sensitive
electrode showed that reaction of NO with molecular oxygen followed
second order kinetics with respect to NO. For the overall reaction we
obtained a third order rate constant of 9.2 10
M
s
, which is in
good accordance to kinetic data published
previously(22, 29, 30) . Determination of an
NO:oxygen stoichiometry close to 4:1 confirms that NO autoxidation may
follow a reaction sequence suggested recently(31) :
The major finding of the present study is that fairly low
concentrations of the NOS cofactor Hbiopterin induce rapid
oxidation of NO. Mechanism of H
biopterin-mediated NO
oxidation is apparently different from that of NO autoxidation, because
it followed zero order instead of second-order kinetics with respect to
NO. Thus, the rate-limiting reaction is independent of the actual NO
concentrations and appears to solely involve H
biopterin and
oxygen. Autoxidation of H
biopterin is thought to generate
superoxide(32) , which again rapidly reacts with NO to form
peroxynitrite(3) . Several observations led us to suggest that
the effect of H
biopterin is mediated by superoxide anions.
First, SOD completely blocked H
biopterin-induced oxidation
of NO. Second, rate constants for NO oxidation in the presence of the
pteridine correlated well with rates of H
biopterin
autoxidation, and, finally, a superoxide generating system was found to
mimic the effects of H
biopterin on NO oxidation kinetics
(see Fig. 4).
Accordingly, superoxide generated during
autoxidation of Hbiopterin seems to react with NO to
peroxynitrite, explaining the rapid, apparently zero order decrease in
NO concentrations induced by H
biopterin. The fact that the
H
biopterin-induced inactivation of NO was not apparent when
either NOS-catalyzed NO production (28) or authentic NO (this
study; not shown) were measured as loss of oxyhemoglobin may be
explained by recent findings demonstrating NO-like reaction of
peroxynitrite with oxyhemoglobin(33) . Thus, it is likely that
peroxynitrite is produced from NO in the presence of
H
biopterin, but direct demonstration of this was precluded
by interference of the reduced biopterin with several photometrical
assays for peroxynitrite (34) (not shown).
Our data suggest
that Hbiopterin reacts with oxygen to yield
5,6-H
biopterin (q-H
biopterin) and
superoxide. If not efficiently removed by SOD, superoxide may combine
rapidly with NO to peroxynitrite. In the presence of reducing compounds
such as thiols or NADPH, q-H
biopterin is
non-enzymatically reduced back to H
biopterin,
presumably explaining the apparent substoichiometrical activity
of the pteridine on NO oxidation under these conditions. Since it is
likely that generated peroxynitrite reacts with H
biopterin
or one of its metabolites, the system consisting of NO, oxygen, and
H
biopterin may be highly complex, and the pertinent
reactions deserve detailed investigation.
The experiments with
purified soluble guanylyl cyclase demonstrate that the products of
Hbiopterin-induced NO oxidation do not stimulate cGMP
formation. In fact, H
biopterin proved to be a potent
inhibitor of the NO-stimulated enzyme. Inhibition was observed between
0.1 and 10 µM H
biopterin, which is in the
range of biopterin concentrations determined in tissue extracts (35) , suggesting that intact cells may express protective
mechanisms preventing NO oxidation and/or convert peroxynitrite to a
biologically active product. In support of latter hypothesis, evidence
has been provided recently that peroxynitrite may act as vasodilator
and inhibit platelet aggregation(36, 37) .
We have
previously demonstrated that purified brain NOS, not saturated with
Hbiopterin, generates hydrogen
peroxide(38, 39) , a reaction that involves production
of superoxide anions as intermediates (40) . Since addition of
exogenous H
biopterin also leads to superoxide generation,
pteridine-deficient NOS may produce sufficient superoxide for NO
inactivation both in the absence and presence of added
H
biopterin, and this is supported by the experiments shown
in Fig. 6. Thus, at low concentrations of H
biopterin
(
1 µM), NOS generates superoxide due to uncoupled
oxygen activation(38) , whereas saturating concentrations of
the pteridine, which prevent the enzymatic oxygen reduction, produce
superoxide non-enzymatically. In each case, the amounts of generated
superoxide are apparently sufficient for complete inactivation of NO,
although we cannot exclude at this stage that another, as yet
unidentified, source of oxygen radicals has contributed to NO
inactivation under the experimental conditions used for this study.
Using NO-chemiluminescence and co-incubation with purified soluble
guanylyl cylcase for detection of NO we have previously observed NO
formation by purified brain NOS even in the absence (guanylyl cylcase
assay) or presence of as little as 30 units/ml SOD (chemiluminescence) (24) . Of note, in this previous study Hbiopterin
concentration-response curves showed sharp maxima at sub-saturating
concentrations of added H
biopterin (0.3-1
µM) in the chemiluminescence and cGMP assays but not in
the citrulline assay. Accordingly, purified NOS may generate small
amounts of free NO at low concentrations of added
H
biopterin, and NO chemiluminescence as well as bioassays
may be considerably more sensitive for NO than Clark-type electrodes.
Our data explain the conflicting reports on feedback inhibition of
NOS (12, 13, 14, 15, 16, 17) ,
if the crude preparations used by others contained predominantly the
Hbiopterin-saturated holoenzyme and low concentrations of
the free pteridine. When we blocked H
biopterin-induced
oxidation of NO by addition of high concentrations of SOD,
NOS-catalyzed citrulline formation was inhibited by 50%, and further
increasing the NO concentrations by SIN-1 produced inhibition down to
about 25% of controls. Lack of effect of SOD in the presence of the NO
scavenger oxyhemoglobin indicates that the effect of SOD was indeed
mediated by NO.
According to these data, NO could inhibit its own
synthesis via interaction with the prosthetic heme group of NOS, but
then it seems hard to understand how the enzyme works. For several
reasons we think that the key in understanding NOS function is
understanding the role of Hbiopterin. NOS is unique as a
pteridine-dependent cytochrome P-450. There must have been compelling
reasons for evolution of H
biopterin as essential cofactor
of NOS, especially in the light of the present data showing that the
pteridine and NO hardly co-exist in the presence of oxygen. Recently,
we compared binding of various pteridine derivatives with their effects
on NOS activity and found that only tetrahydrobiopterins supported
catalytic activity(41) . Thus, the role of
H
biopterin may not be confined to an allosteric effect on
NOS(42, 43) , but additionally involve a redox
reaction. Considering the odd electron chemistry requisite for
formation of the NO radical, it is tempting to speculate that the
heme-catalyzed reaction rather yields NO
or
NO
(44) , and that H
biopterin or q-H
biopterin are required for the eventual
formation and release of NO. Such a function of H
biopterin
would be in accordance with all experimental data available so far,
including previous observations that the pteridine is not involved in
NOS-catalyzed activation of molecular oxygen(38, 39) .
While this manuscript was under consideration, it has been reported
that Hbiopterin markedly diminished inhibition of rat
cerebellar NOS by NO(45) . Griscavage et al.(45) claim that the effects of H
biopterin they
have observed are not due to a direct chemical interaction between the
pteridine and NO. Nevertheless, H
biopterin-induced
oxidation of NO as described in the present study provides a reliable
explanation for virtually all of their data.
This work is dedicated to Prof. Dr. Eycke Böhme(1943-1993).