(Received for publication, February 27, 1995; and in revised form, May 1, 1995)
From the
Peroxynitrite (ONOO
Stimulation of soluble guanylyl cyclase (GTP pyrophosphate-lyase
(cyclizing), EC 4.6.1.2; sGC)
As a free radical, NO reacts with several
intracellular targets(11) . Reaction with molecular oxygen
results in the generation of N
Recent reports indicate that
ONOO
Figure 1:
Effects
of ONOO
Figure 2:
Effects of CDNB (A) and DEM (B) on cGMP accumulation and levels of soluble thiols in
endothelial cells. Endothelial cells were preincubated in culture
medium at 37 °C for 30 min in the presence of increasing
concentrations of CDNB, DEM, or vehicle, washed, equilibrated for 15
min in incubation buffer (see ``Experimental Procedures''),
and stimulated for 4 min with 1 mM ONOO
Figure 3:
Effect of GSH on stimulation of sGC by
ONOO
The biphasic effect of ONOO
Figure 4:
Formation of GSNO from ONOO
Figure 5:
GSH-induced release of NO from GSNO. NO
was measured with a Clark-type NO-sensitive electrode at ambient
temperature in 1.8 ml of a 100 mM sodium phosphate buffer, pH
7.5, in the absence (--) or presence(- - -) of 10 mM EDTA as described under ``Experimental Procedures.'' At
time point zero, a stock solution of GSNO was injected through a septum
to give a final concentration of 50 µM. After 2 min, GSH
and, where indicated, CuCl
Figure 6:
Effect of GSH on stimulation of sGC by
GSNO. Stimulation of purified sGC by authentic GSNO was assayed in the
absence (filledcircles) and presence (unfilledcircles) of 2 mM GSH as described under
``Experimental Procedures.'' Data represent mean values
± S.E. of five separate experiments performed in
duplicate.
Previous reports have demonstrated that ONOO
In good accordance to the results
obtained with intact cells, we found that addition of GSH was not
required for direct stimulation of purified sGC with the NO donor
DEA/NO but obligatory for the effect of ONOO
In spite of
the rather low yield, GSNO formation appears to fully account for
ONOO
Pure GSNO was
stable over a wide range of pH values, but GSH induced a decomposition
of the nitrosothiol that was accompanied by release of NO. These
findings may, at least partially, explain earlier reports on
tissue-induced NO release from GSNO(51, 52) . The
effect of GSH may be mediated by trace metals, since both GSNO
decomposition and NO release were completely inhibited by EDTA, and
accelerated upon addition of CuCl
In keeping with its pronounced effect on NO release from GSNO, GSH
markedly enhanced the potency of the nitrosothiol to stimulate sGC.
Maximal effects of GSNO were smaller than those of DEA/NO, hinting at
secondary reactions limiting the steady state concentrations of free NO
under these conditions. Several mechanisms may account for the apparent
thiol-independent effect of the nitrosothiol. First, it is possible
that the parent compound interacts directly with the prosthetic heme
group of sGC, resulting in enzyme activation. Second, NO may be
released subsequent to trans-nitrosation (56) of free
SH-groups of sGC as recently reported to occur with serum
albumin(57) . Third, the endogenous GSH content of the enzyme
preparations (
The biological
relevance of these findings remains to be established. Although low
yields of GSNO might indicate that ONOO
We thank Dr. Harold F. Hodson, Wellcome Research
Laboratories, Beckenham, United Kingdom, for kindly furnishing S-nitrosoglutathione. We gratefully acknowledge the excellent
technical assistance of Eva Leopold, Margit Rehn, and Jürgen
Malkewitz.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) is widely recognized as
mediator of NO toxicity, but recent studies have indicated that this
compound may also have physiological activity and induce vascular
relaxation as well as inhibition of platelet aggregation. We found that
ONOO
induced a pronounced increase in endothelial
cyclic GMP levels, and that this effect was significantly attenuated by
pretreatment of the cells with GSH-depleting agents. In the presence of
2 mM GSH, ONOO
stimulated purified soluble
guanylyl cyclase with a half-maximally effective concentration of about
20 µM. In contrast to the NO donor
2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO),
ONOO
was completely inactive in the absence of GSH,
indicating that thiol-mediated bioactivation of ONOO
is involved in enzyme stimulation. Studies on the reaction
between ONOO
and GSH revealed that about 1% of
ONOO
was non-enzymatically converted to S-nitrosoglutathione. The authentic nitrosothiol was found to
be stable in solution, but slowly decomposed in the presence of GSH.
GSH-induced decomposition of S-nitrosoglutathione was
apparently catalyzed by trace metals and was accompanied by a sustained
release of NO and a 40-100-fold increase in its potency to
stimulate purified soluble guanylyl cyclase. Our data suggest that the
biologic activity of ONOO
involves S-nitrosation of cellular thiols resulting in NO-mediated
cyclic GMP accumulation.
(
)by L-arginine-derived NO results in intracellular accumulation of
the second messenger cyclic GMP (cGMP) and represents a widespread
signal transduction mechanism involved in a variety of biological
processes, such as endothelium-dependent relaxation, platelet
aggregation, and neurotransmission(1, 2, 3) .
Purification of sGC revealed that the enzyme is a heterodimer
containing stoichiometric amounts of ferro-protoporphyrin
IX(4, 5, 6, 7) . NO exhibits high
affinity for ferrous heme(8) , and a heme-deficient mutant of
sGC has recently been shown to be insensitive to NO(9) ,
supporting the hypothesis that NO activates the enzyme through binding
to the prosthetic heme group resulting in formation of a
ferrous-nitrosyl-heme complex and consequent change in protein
conformation(10) .
O
or other
nitrogen oxides (NO)
with potential cytotoxic
properties(12, 13) . This reaction follows second
order kinetics with respect to NO and is thus rather slow at low NO
concentrations(14, 15, 16) , suggesting that
additional reactions may account for the short half-life of NO in
tissues. Enhanced biological activity of NO in the presence of SOD has
indicated that superoxide contributes to the inactivation of NO (17) due to a fast reaction of NO with
O
to yield peroxynitrite
(ONOO
)(18) . ONOO
is stable
in alkaline solutions, but a species with hydroxyl radical-like
properties is generated as intermediate upon protonation of
ONOO
to the corresponding peroxynitrous
acid(19) . The intermediate appears to be highly reactive,
inducing oxidation of various cellular targets including sulfhydryls (20) and lipids (21, 22, 23) . More
recently, it was demonstrated that ONOO
apparently
mediates several effects previously attributed to NO, e.g. covalent modification of glyceraldehyde-phosphate dehydrogenase (24) and inhibition of aconitase(25, 26) .
Together with the finding that activated macrophages release most of
their NO as ONOO
(27) , these results suggest
that decomposition of ONOO
at physiological pH may be
a major component of NO cytotoxicity.
also may have beneficial effects. Solutions of
authentic ONOO
were shown to induce relaxation of
vascular smooth muscle (28, 29) and to inhibit
platelet aggregation(30) . The molecular mechanisms underlying
these physiological effects of ONOO
are unclear. Liu et al.(28) speculated that relaxation may be due to
small amounts of NO spontaneously released during ONOO
decomposition, and Wu et al.(29) reported on
pronounced increases in NO release when ONOO
was
incubated in the presence of GSH or tissue homogenates. Similarly, Moro et al.(30) found that ONOO
inhibited aggregation of blood platelets only when serum albumin
or GSH were present. It is conceivable, therefore, that the NO-like
properties of ONOO
are due to S-nitrosation
of cellular proteins or GSH. However, the nitrosating potential of
ONOO
is being discussed controversially. Wink et
al.(31) failed to detect an S-nitrosated species
arising from the reaction of ONOO
with GSH, whereas
others have reported on formation of GSNO (30) or another, as
yet unidentified S-nitroso compound(29) . The present
study was designed to elucidate the molecular mechanisms underlying the
biologic activity of ONOO
.
Materials
Recombinant bovine lung soluble
guanylyl cyclase was purified from baculovirus-infected Sf9 cells as
described previously(5, 9) . ONOO was synthesized as described (32, 33, 34) . For inactivation,
ONOO
(10 mM) was incubated for 5 min in 0.5 M phosphate buffer, pH 7.5. GSNO was a kind gift from Dr.
Harold F. Hodson, Wellcome Research Laboratories, Beckenham, United
Kingdom. 2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO) (35) was from NCI Chemical Carcinogen Repository, Kansas City,
MO. 10-fold concentrated stock solutions of DEA/NO were prepared in 10
mM NaOH. [
-
P]GTP (400 Ci/mmol) was
purchased from MedPro (Amersham), Vienna, Austria; the other chemicals
were from Sigma, Vienna, Austria.
Culture of Endothelial Cells and Determination of
Intracellular cGMP
Porcine aortic endothelial cells were
cultured as described previously(36, 37) . Briefly,
endothelial cells were isolated by enzymatic treatment (0.1%
collagenase) and cultured up to 3 passages in Opti-MEM (Life
Technologies, Inc., Vienna, Austria) containing 3% fetal calf serum and
antibiotics. Prior to experiments, endothelial cells were subcultured
in 24-well plastic plates and grown to confluence (2
10
cells/dish). For depletion of GSH, cells were incubated
for 30 min at 37 °C in the presence of different concentrations of
CDNB or DEM prior to experiments. Subsequently, the culture medium was
removed, and the cells were washed once and equilibrated in incubation
buffer (isotonic 50 mM HEPES buffer, pH 7.4, containing 2.5
mM CaCl
, 1 mM MgCl
, 1
mM 3-isobutyl-1-methylxanthine, and 1 µM
indomethacin). After 15 min, ONOO
or DEA/NO were
added to give the initial final concentrations as indicated, and
reactions were stopped 4 min later by removal of the incubation buffer
and treatment for 1 h with 1 ml of 0.01 N HCl. Intracellular
cGMP was measured in the supernatants of the lysed cells by
radioimmunoassay.
Determination of Endothelial GSH Levels
The DTNB
colorimetric method (38) was used for determination of GSH,
which represents the only measurable soluble thiol in endothelial
cells(39) . Cells were cultured in Petri dishes (diameter 90
mm), and confluent monolayers (5
10
cells/dish) were preincubated in culture medium at 37 °C for
30 min in the presence of increasing concentrations of thiol-depleting
agents or vehicle (0.1% ethanol). Cells were washed and equilibrated
for 15 min in incubation buffer (see above). Subsequent to aspiration
of the supernatant, 1 ml of a 2% (w/v) solution of 5-sulfosalicylic
acid was added for cell lysis and deproteinization. Samples were
centrifuged and aliquots of 0.5 ml mixed with 0.5 ml of a 300 mM sodium phosphate buffer, pH 7.5, containing 10 mM EDTA
and 0.2 mM DTNB. After 5 min, the absorbance at 412 nm was
measured and the amount of soluble thiols quantitated by comparison
with GSH standards. The extinction coefficient of the chromophore was
14.3 mM
cm
under these
conditions, which is closely similar to the reported value of 13.6
mM
cm
(38) .
Determination of Guanylyl Cyclase
Activity
Purified soluble guanylyl cyclase (0.15 µg) was
incubated at 37 °C for 10 min in a total volume of 0.2 ml of a
triethanolamine/HCl buffer (50 mM, pH 7.5) containing 0.5
mM [-
P]GTP (200,000-300,000
cpm), 3 mM MgCl
, and 1 mM cGMP in the
absence and presence of 2 mM GSH. Reactions were started by
addition of 20-fold stock solutions of ONOO
, DEA/NO,
or GSNO and stopped by ZnCO
precipitation followed by
isolation of [
P]cGMP by column chromatography as
described(40) . In some experiments, sGC was preincubated for 2
min at 37 °C in the presence of 2 mM GSH and different
concentrations of ONOO
prior to further stimulation
of the enzyme for 8 min with 10 µM DEA/NO in a total
incubation volume of 0.2 ml. Results were corrected for
enzyme-deficient blanks and recovery of cGMP.
Reaction of ONOO
For analysis of S-nitrosated compounds derived
from the reaction of ONOOwith
GSH
with GSH, the authentic
compounds (1 mM each) were incubated for 2 min in 100 mM phosphate buffer at pH values ranging from 6.0 to 8.5, and
30-µl samples were injected onto a 250
4-mm C
reversed-phase column equipped with a 4
4-mm C
precolumn for HPLC analysis (LiChroGraph L 6200, LiChrospher 100
RP-18, 5-µm particle size, Merck). Elution was performed
isocratically at a flow rate of 0.75 ml/min with 20 mM sodium
phosphate buffer, pH 7.4, containing 5% (v/v) methanol. Absorbance was
continuously monitored at 338 nm (LiChroGraph L 4250, Merck) to detect S-nitrosated products. The method was calibrated daily with
freshly prepared solutions of authentic GSNO. Identification of the
reaction product was based on coelution with authentic GSNO in the
following solvents: (i) 20 mM sodium phosphate buffer, pH 3.0,
5% (v/v) methanol; (ii) 20 mM sodium phosphate buffer, pH 7.4;
(iii) 20 mM sodium phosphate buffer, pH 7.4, 1% (v/v)
methanol; (iv) 20 mM sodium phosphate buffer, pH 7.4, 5% (v/v)
methanol; and (v) acetonitrile, H
O, acetic acid
(2.5:97.5:0.1, v/v), yielding retention times of 6.3, 4.4, 4.1, 3.3,
and 2.2 min, respectively. Solvent v has been used by Wu et al. for analysis of S-nitrosated products of
GSH(29) .
GSH-induced Release of NO from GSNO
Stability of
GSNO was assessed spectroscopically with a diode array
spectrophotometer (8452A, Hewlett Packard, Vienna, Austria) in the
absence and presence of 1 mM GSH. Release of NO was measured
aerobically with a commercially available Clark-type electrode (Iso-NO,
World Precision Instruments, Mauer, Germany) as described
previously(16) . Samples were injected through a septum into
completely filled vials to avoid interphase mass transfer. Calibration
of the electrode was performed daily and showed linear response from 10
nM to 1 µM NO with an average slope of 0.8 nM NO/pA output current.
Effects of ONOO
As shown in Fig. 1, ONOOand DEA/NO on
Endothelial cGMP Accumulation
increased cGMP levels in
cultured endothelial cells in a concentration-dependent manner with an
EC
of about 0.2 mM. Maximal effects were
comparable to those elicited by the NO donor DEA/NO, which raised
intracellular cGMP levels up to
50 pmol/10
cells with
an EC
of 0.2 µM. Inactivated ONOO
(1 mM) induced only marginal accumulation of cGMP (from
2.4 ± 0.8 to 4.7 ± 1.1 pmol/10
cells; n = 3). Control experiments performed with NaNO
showed that 0.1 and 1 mM nitrite enhanced endothelial
cGMP levels to 3.1 ± 0.28 and 8.1 ± 1.04 pmol/10
cells (n = 3), respectively, indicating that
nitrite, which was present in the ONOO
stock
solutions at about equimolar concentration, was responsible for the
marginal effect of inactivated ONOO
.
and DEA/NO on cGMP accumulation in
endothelial cells. Endothelial cells were washed, equlibrated for 15
min at 37 °C in incubation buffer (see ``Experimental
Procedures''), and stimulated for 4 min with the indicated
concentrations of ONOO
(filled circles) or
DEA/NO (unfilled circles). Subsequent to aspiration of the
supernatant, cells were lysed with HCl, and intracellular cGMP
accumulation was measured by radioimmunoassay. Data represent mean
values ± S.E. of six separate experiments performed in
triplicate.
To
investigate whether intracellular GSH mediates
ONOO-induced cGMP accumulation, cells were pretreated
for 30 min with increasing concentrations of the GSH-depleting agents
CDNB or DEM(41) . Fig. 2A shows
that CDNB (1-100 µM) decreased endothelial GSH
levels in a concentration-dependent manner and significantly reduced
maximal peroxynitrite-induced cGMP accumulation, whereas the effect of
the NO donor DEA/NO remained unaffected. As shown in Fig. 2B, DEM had very similar effects, albeit at higher
concentrations. At the used concentrations, neither of the two agents
did induce any detectable release of lactate dehydrogenase into the
culture medium (not shown). The GSH levels measured under control
conditions (
10 nmol/10
cells) are in excellent
accordance with values reported previously for cultured endothelial
cells(42) . Based on an endothelial cell volume of 1
pl(43) , this corresponds to intracellular GSH concentrations
of approximately 10 mM.
(unfilledcircles) or 1 µM DEA/NO (filledcircles). Subsequent to aspiration of the
supernatant, cells were lysed with HCl and intracellular cGMP
accumulation was measured by radioimmunoassay. Soluble thiols (stripedcolumns) were determined subsequent to
preincubation of the cells with the indicated drugs by the DTNB
colorimetric method as described under ``Experimental
Procedures.'' Data represent mean values ± S.E. of three
separate experiments performed in
triplicate.
Effects of GSH on Stimulation of sGC by
ONOO
Subsequently, we have
performed experiments with purified sGC to get insights into the
mechanisms accounting for ONOOand DEA/NO
-induced cGMP
accumulation. Incubations have been performed in the absence and
presence of 2 mM exogenously added GSH, but it should be noted
that sGC preparations contained endogenous GSH for stabilization of the
enzyme during purification(5) . However, final dilutions of sGC
resulted in GSH concentrations of less then 10 µM during
incubations. Basal sGC activities were 40.0 ± 13.2 and 78.8
± 15.8 nmol of cGMP
mg
min
(mean ± S.E.; n = 11) in
the absence and presence of 2 mM GSH, respectively. As shown
in Fig. 3A, ONOO
had no effect
on cGMP formation at concentrations of up to 1 mM in the
absence of the added thiol, but markedly stimulated the enzyme in the
presence of 2 mM GSH. The effect was biphasic with a sharp
maximum at 0.1 mM and an EC
of approximately 20
µM ONOO
. The sGC activities maximally
achievable with ONOO
were about 650 nmol cGMP
mg
min
and thus
considerably lower than those observed in the presence of DEA/NO
(
4 µmol
mg
min
) or GSNO (
1 µmol
mg
min
) (see below). The
effect of ONOO
was virtually abolished (1.5-fold
stimulation) when it had been inactivated by incubation for 5 min at pH
7.5 prior to experiments. As observed with endothelial cells, this
marginal stimulation of sGC was mimicked by authentic nitrite, which
induced a 1.6-fold stimulation of the enzyme at a concentration of 0.1
mM (not shown).
and DEA/NO. Stimulation of purified sGC by
ONOO
(A) and DEA/NO (B) was assayed
in the absence (filled circles) and presence (unfilledcircles) of 2 mM GSH as described under
``Experimental Procedures.'' Data represent mean values
± S.E. of three separate experiments performed in
duplicate.
It has been suggested that protein
SH-groups may be critically involved in the regulation of
sGC(44) , indicating that the effect of GSH on
ONOO-induced enzyme stimulation could reflect
reduction of protein sulfhydryls essential for sGC stimulation. To
address this issue, we have additionally studied the effect of GSH on
cGMP accumulation induced by the spontaneous NO donor DEA/NO. Fig. 3B shows that DEA/NO potently stimulated purified
sGC with an EC
of about 50 nM even in the absence
of GSH. Addition of GSH (2 mM) did not potentiate the effect
of DEA/NO but, instead, induced a rightward shift of the concentration
response curve resulting in an EC
of DEA/NO of about 0.3
µM without affecting maximal guanylyl cyclase activity.
These data demonstrate that ONOO
-induced but not
NO-induced stimulation of sGC requires presence of GSH, suggesting that
the effect of the thiol is related to bioactivation of
ONOO
.
and the incomplete activation of sGC indicated that the enzyme
may be inhibited or inactivated by high concentrations of
ONOO
. To investigate this, we have preincubated sGC
for 2 min with increasing concentrations of ONOO
prior to addition of DEA/NO (10 µM). TableI shows that pretreatment of non-stimulated sGC
with high concentrations of ONOO
(
0.1
mM) antagonized further activation of the enzyme by NO. Taking
into account the short half-life (
1 s) of ONOO
at physiological pH(18) , 2 min of preincubation should
have resulted in virtually complete inactivation of
ONOO
, excluding the possibility that the inhibitory
effects were due to a reaction of ONOO
with free NO,
as we have observed recently using a Clark-type electrode for NO
detection.
(
)
Formation, Stability, and Biologic Activity
of GSNO
Reaction of ONOO with GSH (1 mM each) was complete within 2 min and resulted in formation of a
compound, which exhibited a light absorbance maximum at 338 nm and
co-eluted with authentic GSNO from RP-18 columns in five different
solvents. Fig. 4shows that formation of GSNO
was increased at increasing pH; half-maximal efficiency was observed at
pH 7.0, maximal yields required pH values
8.5. The reaction was
not inhibited in the presence of EDTA (not shown).
and GSH as a function of pH. Authentic ONOO
(1
mM) was allowed to react with GSH (1 mM) for 2 min at
ambient temperature in 100 mM phosphate buffer adjusted to pH
values as indicated. Samples (30 µl) were analyzed for GSNO by HPLC
as described under ``Experimental Procedures.'' Data
represent mean values ± S.E. of three separate
experiments.
If this
non-enzymatic S-nitrosation of GSH was responsible for the
cGMP accumulation induced by ONOO, GSNO should
decompose and release NO under certain conditions. We have investigated
this issue by both spectrophotometric analysis of authentic GSNO and
electrochemical measurement of NO release. As revealed by recording the
absorbance at 338 nm over time, GSNO was stable for at least 5 h at pH
2.0-9.0. However, presence of 1 mM GSH induced a
time-dependent, EDTA-sensitive decomposition of the nitrosothiol (t
3 h at pH 7.5; not shown). Decomposition of GSNO was
accompanied by release of NO as revealed by the electrochemical
measurements shown in Fig. 5. GSNO (50 µM)
alone induced a very slight transient response of a Clark-type NO
electrode, corresponding to apparent NO concentrations of less than 20
nM, whereas a pronounced, long-lasting release of NO was
observed upon addition of 50 µM GSH. GSH-induced NO
release was markedly enhanced in the presence of 10 µM CuCl
and completely blocked by 10 mM EDTA.
The initial rates of NO release shown in Fig. 5were 0.46 and
2.00 µM min
in the absence and presence
of added CuCl
, respectively, steady state concentrations of
NO were reached about 2 min after addition of GSH.
where added to give final
concentrations of 50 and 10 µM, respectively. An original
tracing representative for three similar experiments is
shown.
GSH-induced
release of NO from GSNO was further confirmed in experiments with
purified sGC. Fig. 6shows that GSNO stimulated the
formation of cGMP in a concentration-dependent manner. Maximal sGC
activities of about 1 µmol of cGMP mg
min
were observed with 0.3 mM GSNO, the EC
of the nitrosothiol was 8.0
µM. In the presence of 2 mM GSH, the EC
of GSNO was 0.18 µM and maximal effects were
obtained at GSNO concentrations as low as 1 µM, further
suggesting that GSH triggers release of NO from the nitrosothiol.
exhibits NO-like biologic activities, inducing relaxation of
blood vessels (28, 29) and inhibiting platelet
aggregation(30) . The present study strongly suggests that
these effects of ONOO
may be mediated by
thiol-dependent stimulation of sGC. Authentic ONOO
induced a pronounced accumulation of endothelial cGMP levels with
a maximal effect comparable to that of the NO donor DEA/NO. Consistent
with rather long diffusion distances, presumably resulting in
significant decomposition of the added ONOO
(18) , accumulation of cellular cGMP required about
10-fold higher concentrations of ONOO
(EC
0.2 mM) than stimulation of purified soluble
guanylyl cyclase (EC
0.02 mM; see below).
The effect of ONOO
on endothelial cGMP accumulation
was significantly attenuated by depletion of intracellular GSH with the
thiol-depleting agents CDNB and DEM, indicating that GSH is an
important cellular target mediating the biologic activity of
ONOO
. However, the effects of the drugs were
incomplete, and at higher concentrations these agents were toxic to the
cells (not shown). Thus, the residual GSH (approximately 1-2
mM) could be sufficient to support GSNO formation or,
alternatively, additional targets may be involved in ONOO
action. Free sulfhydryl groups of cellular proteins, for
instance, could react with ONOO
to yield comparably
stable S-nitroso derivatives that slowly release
NO(45, 46) . Lack of effect of CDNB and DEM on cGMP
accumulation induced by DEA/NO is in accordance with previous findings
showing that the effects of several NO donors were not significantly
affected by pretreatment of endothelial cells with thiol-depleting
drugs(39, 47, 48) . Taken together, these
data indicate that NO-induced accumulation of cGMP occurs in a
thiol-independent manner, whereas the effect of ONOO
may involve modification of intracellular sulfhydryls and perhaps
additional as yet unknown targets.
.
Although presence of low concentrations of endogenous GSH did not allow
us to unambiguously demonstrate thiol-independent enzyme stimulation,
our results are consistent with a previous study investigating this
issue using thiol-free sGC that had been purified under anaerobic
conditions(49) . With an EC
of about 50
nM, DEA/NO is the most potent NO donor described so far. For
unknown reasons, DEA/NO was somewhat less potent in the presence of
GSH. Under these conditions, the drug exhibited an EC
of
about 0.3 µM, which is virtually the same as that found
for DEA/NO-induced endothelial cGMP accumulation (0.2 µM;
this study) and vascular relaxation (0.2 µM)(35) .
In contrast to DEA/NO, ONOO
stimulated purified sGC
in a strictly GSH-dependent manner. The maximal effects of
ONOO
were much less pronounced than those of DEA/NO,
apparently due to the inactivation of sGC by high concentrations of
ONOO
, revealed by the biphasic effect shown in Fig. 3A and inhibition of sGC by preincubation with
high concentrations of ONOO
(Table 1).
Our
data suggest that the biologic activity of ONOO may
be mediated by non-enzymatic nitrosation of GSH. A previous
investigation of ONOO
-induced sulfhydryl oxidation
points to the formation of the corresponding sulfenic or sulfinic
acids(20) , but formation of nitrosothiols has not been
investigated in this earlier study. Recently, Wink et al.(31) have reported lack of GSNO formation from
ONOO
reacting with GSH, but the applied photometrical
method is probably not sufficiently sensitive to allow detection of
small amounts of S-nitrosothiols. Others have reported on
formation of an S-nitroso derivative of GSH being distinct
from GSNO(29) , which we have never observed in the course of
product analysis by HPLC. However, in accordance with a recent study by
Moro et al.(30) , we found that about 1% of the added
ONOO
reacted with GSH to yield an S-nitrosylated compound, which has been identified as GSNO
based on co-elution with the authentic nitrosothiol from a reversed
phase HPLC column in five different solvents. The reaction mechanisms
involved in ONOO
-induced S-nitrosation of
thiols is still elusive. The pH dependence of GSNO formation is in good
accordance with previous results on ONOO
-induced
sulfhydryl oxidation(20) , suggesting that the reactive species
is peroxynitrite anion, rather than peroxynitrous acid. However, direct
reaction of ONOO
with GSH to yield GSNO would result
in concomitant release of peroxide anion and seems unlikely. Since only
1% of the added ONOO
was converted to GSNO, it
is conceivable that other reactive metabolites occurring in the course
of sulfhydryl oxidation by ONOO
(20) are also
involved in S-nitrosation. Even though EDTA did not block GSNO
formation, it cannot be ruled out that the reaction is catalyzed by
trace metals, since the catalytic activity of transition metals is not
always prevented by chelating compounds(50) .
-induced stimulation of purified sGC. According
to the data shown in Fig. 4, presence of 0.1 mM ONOO
should have resulted in formation of 0.6
µM GSNO at pH 7.5, and this is apparently sufficient for
the observed stimulation of sGC (see Figs. 3A and 6). Further
stimulation of cGMP formation is probably antagonized by inactivation
of the enzyme in the presence of high concentrations of
ONOO
. This inhibitory effect of
0.1 mM ONOO
could either be due to oxidation of
critical amino acid residues or involve oxidative modification of the
prosthetic heme group by reactive ONOO
metabolites,
resulting in decreased sensitivity of sGC toward NO.
(see Fig. 5). The
effect of copper ions may be direct or involve GSH peroxides as
suggested previously for thiol-mediated oxidation of low density
lipoprotein(53) . Interestingly, the effect of GSH was mimicked
by ascorbate (not shown), indicating that GSNO decomposition may be
catalyzed by copper(I) ions and that GSH or other reducing agents are
required for reduction of the added copper(II) ions (50) .
These results are in accordance with a previous report on metal
ion-catalyzed decomposition of the nitrosothiol S-nitroso-N-acetyl-DL-penicillamine (54) and with a recent study demonstrating that chelation of
copper(I) ions reduces the biologic activity of GSNO(55) .
10 µM) may have been sufficiently high
to induce release of NO. Finally, enzyme stimulation could be due to
the very minor amounts of NO that are apparently released from GSNO
even in the absence of GSH (see Fig. 5).
-mediated
nitrosation is insignificant in cells, it is conceivable that the
reaction becomes more efficient when ONOO
is
continuously generated instead of being added as concentrated stock
solution. Furthermore, in intact cells, cGMP levels were increased to
the same maximal values by ONOO
and DEA/NO, whereas
stimulation of purified sGC by ONOO
was only
15-20% of that achieved with the NO donor, indicating that
activation of ONOO
through S-nitrosation may
be facilitated in intact cells by specific enzymes or other mechanisms.
If ONOO
should turn out to be the physiological
product of the NO synthase reaction, as suggested by recent studies
with the purified enzyme (16) and agonist-stimulated
endothelial cells(58) , ONOO
-induced S-nitrosation could, in fact, represent a key reaction in
NO/cGMP-mediated signal transduction.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.