From the Institut für Pharmakologie und
Toxikologie, Karl-Franzens-Universität Graz,
Universitätsplatz 2, A-8010 Graz, Austria and the ¶ Institut
für Pharmakologie, Freie Universität Berlin, Thielallee
69-73, D-14195 Berlin, Germany
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ABSTRACT |
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Nitric oxide (NO), a physiologically
important activator of soluble guanylyl cyclase (sGC), is synthesized
from L-arginine and O2 in a reaction
catalyzed by NO synthases (NOS). Previous studies with purified NOS
failed to detect formation of free NO, presumably due to a fast
inactivation of NO by simultaneously produced superoxide (O2).
To characterize the products involved in NOS-induced sGC activation, we
measured the formation of cyclic 3
,5
-guanosine monophosphate (cGMP)
by purified sGC incubated in the absence and presence of GSH (1 mM) with drugs releasing different NO-related species or
with purified neuronal NOS. Basal sGC activity was 0.04 ± 0.01 and 0.19 ± 0.06 µmol of cGMP × mg
1 × min
1 without and with 1 mM GSH, respectively.
The NO donor DEA/NO activated sGC in a GSH-independent manner.
Peroxynitrite had no effect in the absence of GSH but significantly
stimulated the enzyme in the presence of the thiol (3.45 ± 0.60 µmol of cGMP × mg
1 × min
1). The
NO/O
2 donor SIN-1 caused only a slight accumulation of cGMP in
the absence of GSH but was almost as effective as DEA/NO in the
presence of the thiol. The profile of sGC activation by Ca2+/calmodulin-activated NOS resembled that of SIN-1; at a
maximally active concentration of 200 ng/0.1 ml, NOS increased sGC
activity to 1.22 ± 0.12 and 8.51 ± 0.88 µmol of cGMP × mg
1 × min
1 in the absence and presence
of GSH, respectively. The product of NOS and GSH was identified as the
thionitrite GSNO, which activated sGC through Cu+-catalyzed
release of free NO. In contrast to S-nitrosation by peroxynitrite, the novel NO/O
2-triggered pathway was very
efficient (25-45% GSNO) and insensitive to CO2.
Cu+-specific chelators inhibited bradykinin-induced cGMP
release from rat isolated hearts but did not interfere with the direct activation of cardiac sGC, suggesting that thionitrites may occur as
intermediates of NO/cGMP signaling in mammalian tissues.
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INTRODUCTION |
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The NO/cGMP pathway involving NO-mediated activation of soluble guanylyl cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2.; sGC1) is essential to signal transduction in several biological systems (1). In the vasculature, NO/cGMP signaling is important for the regulation of blood pressure and platelet function (2); in the brain, this pathway controls the release of neurotransmitters such as glutamate and acetylcholine (3). Biosynthesis of NO is triggered by autacoids increasing the intracellular concentration of free Ca2+, resulting in activation of Ca2+/calmodulin-dependent NOS (EC 1.14.13.39), complex homodimeric enzymes that catalyze the synthesis of NO from the guanidino moiety of the amino acid L-arginine (4-7). The oxidation of L-arginine is catalyzed by a cytochrome P450-type heme iron in the oxygenase domain of NOS with O2 serving as a cosubstrate. The electrons required for reduction of O2 are shuttled from the cofactor NADPH to the heme via a flavin-containing cytochrome P450 reductase that forms the C-terminal half of the NOS protein. This electron transport chain only operates when Ca2+/calmodulin is bound to the enzyme, which then effects the Ca2+ regulation of endothelial and neuronal NO synthesis.
At low concentrations of L-arginine or in its absence, the
enzymatic reduction of O2 uncouples from substrate
oxidation and results in the generation of superoxide anions and
H2O2 (8-12). The effective coupling of the
reaction requires not only saturation with L-arginine but
also the pteridine cofactor H4biopterin (13). Since the two
subunits of neuronal NOS bind H4biopterin in a highly anticooperative manner, the purified enzyme always contains 1 molecule of H4biopterin/dimer, i.e. it consists
of a H4biopterin-containing and a
H4biopterin-free subunit (14). In this state, the enzyme can form L-citrulline and is stimulated about 2-fold upon
binding of H4biopterin to the low affinity site of the
pteridine-free subunit. Together with our recent findings that the two
NOS subunits function independently (15), this very unusual binding
behavior of H4biopterin appears to have interesting
functional consequences; at low concentrations of free
H4biopterin, the H4biopterin-containing subunit
of NOS is expected to form NO, whereas the uncoupled NADPH oxidation
catalyzed by the pteridine-free subunit should generate O
2,
which reacts at a nearly diffusion-controlled rate (k = 4.3-6.7 × 109 M
1
s
1) with NO to form peroxynitrite, a powerful oxidant and
cytotoxic species (16). In keeping with the hypothesis that
simultaneous production of NO and O
2 is an intrinsic activity
of NOS, the enzyme does not catalyze formation of free NO unless high
concentrations of SOD are present to outcompete the peroxynitrite
reaction (17, 18). The simultaneous production of NO and O
2
could be important to prevent feedback inhibition of the enzyme by free
NO (19). In the presence of saturating concentrations of
H4biopterin, which prevent enzymatic O
2 formation
due to coupling of NADPH and L-arginine oxidation, NO
becomes inactivated by O
2 formed non-enzymatically by
autoxidation of H4biopterin (17, 20). As an alternative explanation of these puzzling results, it has been suggested that the
true product of NOS is NO
(18), which could be converted
to NO by stoichiometric amounts of SOD (21).
NO was shown to be the only redox form of nitrogen monoxide capable of
activating sGC (22), raising the question of how the NO signal is
carried from NOS to sGC. A possible mediator is peroxynitrite acting
via the nitrosation of GSH to GSNO. Although this reaction is
inefficient and yields maximally 1% GSNO (23, 24), there could be as
yet unrecognized more efficient nitrosative pathways accounting for
GSNO biosynthesis in vivo. Alternatively, cells may contain
sufficient SOD to prevent inactivation of NO by O2. As it is
difficult to predict which of these pathways is involved in the true
chemical route of NOS/sGC signaling, we have designed an in
vitro study using a reconstituted system consisting of purified
neuronal NOS and purified sGC. This system allowed us to compare the
action profile of NOS with that of donor compounds of several nitrogen
oxides and to study the activation of sGC by active NOS in the presence
of GSH and SOD. Our study yielded several unexpected results. Most
importantly, the data uncover a novel NO/O
2-triggered
nitrosative pathway that outcompetes the reaction of O
2 with
both NO and SOD.
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EXPERIMENTAL PROCEDURES |
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Materials--
Purified recombinant rat neuronal NOS was
obtained from baculovirus-infected insect cells as described (25). sGC
was purified from bovine lung by immunoaffinity chromatography as
described previously (26). Alkaline stock solutions of peroxynitrite
(80-100 mM) were prepared and quantified as described
(27). L-[2,3,4,5-3H]Arginine hydrochloride
(57 Ci/mmol) and [-32P]GTP (400 Ci/mmol) were from
Amersham, purchased through MedPro (Vienna, Austria). DEA/NO, SPER/NO,
Angeli's salt, and GSNO were obtained from Alexis (Läufelfingen,
Switzerland). SIN-1 was a generous gift from Dr. K. Schönafinger
(Höchst Marion Rousell Inc., Frankfurt, Germany).
H4Biopterin was from Dr. B. Schircks Laboratories (Jona,
Switzerland) and NADPH from Boehringer Mannheim (Vienna, Austria).
Other chemicals including Cu,Zn-SOD (specific activity 4,200 units/mg)
were from Sigma (Vienna, Austria).
Isolated Heart Perfusion--
Rat hearts were isolated and
retrogradely perfused with Krebs-Henseleit bicarbonate buffer at 9.0 ml × min1 × g
1 wet weight as
described previously (28). After equilibration (45 min), hearts were
perfused via side line for 15 min with bradykinin (0.1 µM) or SPER/NO (0.1 mM), followed by
perfusion (15 min) with L-NNA (0.2 mM), the
copper ion-selective chelators neocuproine or BCS (both
Cu+-selective), or cuprizone (Cu2+-selective)
(all chelators at 0.2 mM) in the absence of agonists. Finally, agonists were perfused together with copper chelators or
L-NNA, again over 15 min. Coronary effluent samples were
collected in 3-min intervals during the first and last period of 15 min, i.e. the agonist infusion periods, and stored at
20 °C pending analysis by radioimmunoassay.
Determination of GSNO-- HPLC analysis of GSNO was performed on a C18 reversed phase column with 20 mM phosphate buffer (pH 3.0), containing 20 µM neocuproine to prevent GSNO decomposition, at 1 ml/min and detection at 338 nm as described (24). To remove nitrite, samples were treated with 10 µl of ammonium sulfamate (10 mM) prior to acidification (pH ~3). Calibration curves were established with GSNO under enzyme assay conditions. GSNO was not detectable in samples containing up to 0.1 mM NaNO2.
For a more sensitive determination of GSNO, NO was released by addition of Cu(NO3)2 (29) and quantified with an NO electrode connected to an Apple Macintosh computer via an analog-to-digital (A/D) converter (World Precision Instruments, Mauer, Germany) (17). The output current was recorded at 0.6 Hz under constant stirring at 37 °C in open glass vials. For calibration, Cu(NO3)2 (10 mM final) was added to GSNO under NOS/sGC assay conditions (see below). Linear calibration curves were obtained from plots of the NO versus the GSNO (0.05-5 µM) concentration. GSNO was not detectable in samples containing up to 50 µM NaNO2. As described in detail elsewhere,2 this method is selective for thionitrites but does not allow the specific detection of GSNO in biological samples where sulfhydryls other than GSH may be present. In some experiments, the total concentration of nitrosothiols was determined spectrophotometrically according to the method of Saville (30).Enzyme Assays--
Stock solutions of purified bovine lung sGC
(vmax = ~ 16 µmol of cGMP × mg1 × min
1) were diluted to 250-fold final
concentrations (0.125 mg/ml) with chilled 50 mM potassium
phosphate buffer (pH 7.4) containing 0.5 mg/ml bovine serum albumin.
0.4 µl of the diluted enzyme (50 ng) was added to 89.6 µl of 50 mM potassium phosphate buffer (pH 7.4), containing 0.55 mM [
-32P]GTP (~200,000 cpm), 3.3 mM MgCl2, 1.1 mM cGMP, 0.11 mM L-arginine, 55 µM NADPH, 11 µM CaCl2, and 1.1 µg of calmodulin. GSH,
SOD, drugs, and NaHCO3 were present as indicated. Reactions
were started by addition of 0.5-500 ng of purified NOS
(vmax = 0.7-0.9 µmol of
L-citrulline × mg
1 × min
1) in 10 µl of a chilled 50 mM potassium
phosphate buffer (pH 7.4) containing 0.4 mM CHAPS, followed
by incubation of the samples at 37 °C for 10 min, and isolation of
[32P]cGMP as described (31). Where indicated, sGC was
incubated with donor compounds instead of NOS under identical
conditions.
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RESULTS |
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Effect of GSH on the Activation of sGC by NO Donors and Neuronal
NOS--
To allow a reliable comparison of the effects of NO donors
with that of the enzymatic NOS product(s), determination of sGC activity was performed in 50 mM phosphate buffer (pH 7.4)
containing 0.5 mM GTP, 3 mM MgCl2,
1 mM cGMP, 0.1 mM L-arginine, 50 µM NADPH, 10 µM CaCl2, and 10 µg/ml calmodulin. Under these conditions, NOS activity was 0.08-0.1
µmol of L-citrulline × mg1 × min
1, whether or not GSH (1 mM) was
present.
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Effects of SOD on sGC Activation by NOS--
As shown in Fig.
2A, presence of SOD led to an
apparently GSH-independent activation of sGC by NOS (200 ng/0.1 ml).
The effective concentrations of SOD (EC50 = 0.66 ± 0.13 unit/ml) were about 200-fold lower than those required to detect
free NO under the same conditions (EC50 = 135 ± 7 units/ml; data not shown). Identical data were obtained with MnSOD from
Escherichia coli (data not shown). In the presence of GSH,
SOD increased NOS-stimulated cGMP formation to 13.74 ± 1.28 µmol × mg1 × min
1, a value close
to that obtained with DEA/NO (cf. Fig. 1). As evident from
the concentration-response curves shown in Fig. 2B, the
system was highly sensitive to NOS. In the presence of GSH, the
Ca2+/calmodulin-activated enzyme stimulated sGC with an
EC50 value of 22.5 ± 7.5 ng/0.1 ml (0.7 nM) (filled circles). SOD (unfilled squares) decreased the EC50 of NOS to 2.2 ± 0.6 ng/0.1 ml. The slight increase of basal sGC activity (2.56 ± 0.74 µmol of cGMP × mg
1 × min
1) was
inhibited by hemoglobin and the heme-site inhibitor ODQ (35) (data not
shown), indicating that it was due to stabilization of ambient NO (36).
If SOD and GSH were present together (filled squares), the
resulting EC50 (8.2 ± 1.2 ng of NOS/0.1 ml) was between the values obtained with either SOD or GSH alone.
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Characterization of NOS-induced sGC Activation in the Presence of
GSH--
The surprising difference between sGC activation by
NO/O2 (generated by NOS or SIN-1) and peroxynitrite
(cf. Fig. 1) prompted us to perform further experiments to
better define their respective modes of action and to characterize
possible intermediates of these pathways. Very similar data to those
described below were obtained when sGC was activated with the
NO/O
2 donor SIN-1 instead of
NOS.3
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Physiological Significance of NO/O2-triggered
S-Nitrosation--
S-Nitrosation triggered by peroxynitrite
occurs at low yields (23, 24) and is blocked by CO2
(cf. Figs. 3 and 5), suggesting that it may not represent an
important pathway of NO/cGMP signaling in vivo. However, the
present data show that S-nitrosation by NOS is efficient at
physiologically low enzyme concentrations and not inhibited by
CO2. Thus, the novel NO/O
2-triggered nitrosative pathway described here could be physiologically relevant. To study the
possible contribution of GSNO as an intermediate in the NO/cGMP signal
transduction cascade, we decided to test whether inhibition of
non-enzymatic GSNO decomposition by chelators of copper ions affects
agonist-induced cGMP formation in a perfused isolated rat heart system.
As shown in Fig. 7A, the
endothelium-dependent vasodilator bradykinin stimulated a
NG-nitro-L-arginine
(L-NNA)-sensitive release of cGMP into the coronary effluent, which was strongly inhibited by the Cu+-selective
chelators neocuproine and BCS but not by the Cu2+ chelator
cuprizone (38). To exclude that these effects were due to an
interference of the drugs with sGC stimulation or cGMP outward
transport, similar experiments were performed with the NONOate SPER/NO,
which is expected to activate cardiac sGC directly. Release of cGMP
triggered by the NO donor was not affected by neocuproine or by
cuprizone (Fig. 7B), demonstrating that
Cu+-specific chelators are specific inhibitors of
agonist-induced cGMP release from rat heart.
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DISCUSSION |
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The present study was designed to elucidate the molecular mechanisms involved in cGMP accumulation triggered by Ca2+-activated NOS. Purified sGC was functionally reconstituted with purified neuronal NOS or incubated with donors of nitrogen oxides to learn about the conditions required for the effective transduction of the NOS signal and the identity of the intermediates involved. Special emphasis was given to the role of the sulfhydryl compound GSH, which occurs in concentrations of 1-10 mM in mammalian cells (39). The NO donor DEA/NO stimulated sGC in a GSH-independent manner, demonstrating that the direct activation of the enzyme by NO does not require the presence of a thiol. However, earlier reports appear to show the opposite (40), but sodium nitroprusside or organic nitrates, which were used as NO donors before better drugs became available in the early 1990s, do not release free NO in well defined first order reactions but require chemical transformations to become biologically active.
NOS has never been observed to produce NO unless fairly high amounts of
SOD were present (17, 18). Based on the findings that (i) NOS generates
O2 if not saturated with both L-arginine and
H4biopterin (9, 10), (ii) the enzyme is only half-saturated with H4biopterin over a wide range of exogenous
H4biopterin concentrations due to anticooperative pteridine
binding (14), (iii) the two subunits of NOS dimers function
independently (15), and (iv) autoxidation of exogenous
H4biopterin results in O
2-mediated inactivation of
NO (17), it appears safe to conclude that NOS generates NO and
O
2 simultaneously under most circumstances and that SOD acts
via scavenging O
2. This view is further supported by the
present results showing that the effect of NOS resembled that of the
NO/O
2 donor SIN-1 (33), with a pronounced GSH dependence of
sGC activation that was overcome by addition of SOD. According to an
alternative proposal, NOS produces nitroxyl (NO
), which
is converted to NO by stoichiometric amounts of SOD (18). However, we
observed only minor activation of sGC with Angeli's salt at a
concentration yielding a 10-fold higher rate of product release than
achieved with a maximally active concentration of DEA/NO (1 µM). The small effect that we observed with Angeli's salt may be due to release of minor amounts of NO and/or aerobic conversion of NO
to NO (41). Together with the present
knowledge about NOS enzymology, our results argue against
NO
as the biologically active species produced by NOS,
although we cannot exclude that the enzyme produces an NO
species with different chemical properties than the NO
released from Angeli's salt.
Activation of sGC by NOS was rendered GSH-independent by remarkably low
concentrations of SOD, which are clearly below the average
concentration of SOD in tissues (10 µM corresponding to about 1,000 units/ml) (42). A maximal effect was obtained with 5 units
of SOD/ml, conditions under which enzymatic NO formation is below the
detection limit of the NO
electrode.4 These surprising
results are difficult to explain but may be of utmost physiological
importance because they suggest that low levels of SOD, which are far
below the levels required to outcompete the peroxynitrite reaction, may
be sufficient for the effective functional coupling of NOS activity to
cGMP accumulation. At a first glance, the data also seem to suggest
that the reaction of NO/O2 with GSH is less important because
of the ubiquitous occurrence of SOD in tissues. However, GSH largely
antagonized the pronounced leftward shift of the NOS
concentration-response curve caused by SOD (cf. Fig.
2B), indicating that NO/O
2 preferentially reacts
with the thiol when both GSH and SOD are present. It is interesting
that these major components of the cellular defense machinery against
oxidative stress (43) act synergistically to prevent the formation of
peroxynitrite under conditions of NO/O
2 generation.
It was surprising to discover that significant amounts of GSNO were
formed from NO/O2 produced by NOS (this study) or released from SIN-1.3 Our findings indicate that GSH nitrosation by
NO/O
2 competes effectively with peroxynitrite formation.
Taking into account that NO reacts with O
2 at nearly
diffusion-controlled rates (44), GSNO formation must involve a
comparably rapid reaction. Free NO does not nitrosate thiols at
significant rates (45), but the reaction of NO with O2
results in formation of a potent nitrosating intermediate (46, 47). The
autoxidation reaction is second order with respect to NO and thus too
slow to compete with peroxynitrite formation at submicromolar
concentrations of NO (48-50). According to a recent proposal, however,
S-nitrosation does occur at low NO concentrations with
O2 or other oxidants serving as electron acceptors (51).
Unfortunately, there is no rate constant available for this reaction,
making it difficult to judge whether it could account for our
observations. The reaction of NO with peroxynitrite, which also could
yield a nitrosating species is too slow to be involved in GSNO
formation (27).
As a likely explanation for NO/O2-triggered
S-nitrosation, we propose that thiyl radicals (GS·)
originating from oxidation of GSH by O2,
H2O2, O
2, or peroxynitrite combine
with NO· to form GSNO. It is conceivable that GSH oxidation by
initially formed peroxynitrite is a source of thiyl radicals. Such a
mechanism would predict that maximally 50% of product flow were
recovered as GSNO, a value that agrees well with our observations
(cf. Fig. 6). Based on the finding that GSNO formation was
insensitive to NaHCO3, this proposal has to be dismissed if
the 1-electron oxidation of GSH by peroxynitrite (52, 53) is inhibited
by CO2. Current evidence points to a partial inhibition
(54, 55), but the effect of CO2 on thiyl radical formation
has not been reported. Although the precise mechanism of
NO/O
2-triggered S-nitrosation remains to be
clarified, this pathway may at least partially explain the occurrence
of S-nitrosothiols in mammalian tissues (56-58). These
compounds are relatively insensitive to O2 and O
2
and may serve as a storage or transport form of NO (56, 59, 60). GSNO
can nitrosate thiol groups of proteins and so regulate important physiological variables, e.g. the activity of tissue-type
plasminogen activator (61), the down-regulation of the
N-methyl-D-aspartate receptor (62), and the
O2 affinity of hemoglobin (63).
Our data show that NO and O2 take part in reactions clearly
more diverse than previously known (see scheme in Fig.
8). The key determinants of the link
between NOS and sGC are the levels of SOD and GSH. With less GSH or
SOD, as inferred in certain pathologies (64, 65), NO/O
2 will
exacerbate peroxynitrite-mediated tissue damage (16). Low levels of SOD
would not prevent formation of peroxynitrite but appear to be
sufficient for the coupling of NOS activity to cGMP accumulation.
Whether SOD is present or not, the NO/O
2 pathway appears to be
significantly shifted toward GSNO in the presence of physiologically
occurring concentrations of GSH.
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Since the biological activities of GSNO differ considerably from those
of NO (66), the homolytic cleavage of the thionitrite may represent an
additional regulatory site of NO/cGMP signaling by which the outcome of
NOS activity is switched toward cGMP accumulation, vascular relaxation,
inhibition of platelet function, and synaptic transmission in the
brain. It is well established that GSNO decomposition is catalyzed by
Cu+ ions (29, 38, 67, 68). Although a marked mobilization of redox-active copper occurs in myocardial ischemia/reperfusion injury
(69), the cellular availability of Cu+ is probably limited
under physiological conditions, suggesting that enzymatic pathways of
GSNO decomposition may be more relevant than the non-enzymatic
Cu+ mechanism. Several enzymes including GSH peroxidase
(70, 71), thioredoxin reductase (72), and -glutamyl transpeptidase
(73) appear to catalyze reactions leading to NO release from GSNO. Of
note, a Cu+-dependent enzymatic activity was
reported to catalyze GSNO decomposition in platelets (74). In the
present study, we found that Cu+-selective chelators led to
a pronounced inhibition of bradykinin-induced release of cGMP into the
coronary effluent of isolated perfused rat hearts, whereas cGMP release
upon direct activation of cardiac sGC was not affected by the
chelators. Although these data are good circumstantial evidence that
Cu+-dependent release of NO from endogenous
thionitrites may be essentially involved in cardiac NO/cGMP signaling,
further studies are needed to unequivocally demonstrate the role of
GSNO as a product of the NOS pathway in mammalian tissues.
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ACKNOWLEDGEMENTS |
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We thank Eva Pitters, Gerald Wölkart, Margit Rehn, and Jürgen Malkewitz for excellent technical assistance; Dr. Kim Q. Do (Institute for Brain Research, Zürich, Switzerland) for helpful suggestions on the HPLC analysis of GSNO; and Dr. Benjamin Hemmens for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich (to B. M.) and the Deutsche Forschungsgemeinschaft (to D. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 43-316-380-5567; Fax: 43-316-380-9890; E-mail: mayer{at}kfunigraz.ac.at.
1
The abbreviations used are: sGC, soluble
guanylyl cyclase; BCS, bathocuproine sulfonic acid; CHAPS,
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; DEA/NO,
2,2-diethyl-1-nitroso-oxyhydrazine; GSNO,
S-nitrosoglutathione; H4biopterin,
(6R)-5,6,7,8-tetrahydro-L-biopterin;
L-NNA,
NG-nitro-L-arginine;
NO, nitroxyl anion; NOS, nitric oxide synthase;
SIN-1, 3-(4-morpholinyl)-sydnonimine; SPER/NO, spermine/NO; SOD,
superoxide dismutase; HPLC, high performance liquid
chromatography.
2 S. Pfeiffer, A. Schrammel, K. Schmidt, and B. Mayer, submitted for publication.
3 A. Schrammel, S. Pfeiffer, D. Koesling, K. Schmidt, and B. Mayer, submitted for publication.
4 S. Pfeiffer and B. Mayer, unpublished data.
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REFERENCES |
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