Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels.

Research article
2552 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 9 September 2006
Targeting the heme-oxidized nitric oxide
receptor for selective vasodilatation
of diseased blood vessels
Johannes-Peter Stasch,1 Peter M. Schmidt,1,2 Pavel I. Nedvetsky,3 Tatiana Y. Nedvetskaya,3
Arun Kumar H.S.,3 Sabine Meurer,2,4 Martin Deile,3 Ashraf Taye,3 Andreas Knorr,1
Harald Lapp,5 Helmut Müller,3 Yagmur Turgay,3 Christiane Rothkegel,1,6 Adrian Tersteegen,1
Barbara Kemp-Harper,2,7 Werner Müller-Esterl,4 and Harald H.H.W. Schmidt2,7
1Institute of Cardiovascular Research, Bayer HealthCare, Wuppertal, Germany. 2Department of Pharmacology, Monash University,
Melbourne, Victoria, Australia. 3Rudolf-Buchheim-Institute for Pharmacology, Giessen, Germany. 4Institute for Biochemistry II,
University of Frankfurt Medical School, Frankfurt, Germany. 5Helios Klinikum Erfurt, Erfurt, Germany. 6Martin-Luther-University,
School of Pharmacy, Halle, Germany. 7Centre for Vascular Health, Monash University, Melbourne, Victoria, Australia.
ROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/
cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strong oxidant
peroxynitrite. Increased oxidative stress affects the heme-containing NO receptor sGC by both decreasing
its expression levels and impairing NO-induced activation, making vasodilator therapy with NO donors less
effective. Here we show in vivo that oxidative stress and related vascular disease states, including human
diabetes mellitus, led to an sGC that was indistinguishable from the in vitro oxidized/heme-free enzyme.
This sGC variant represents what we believe to be a novel cGMP signaling entity that is unresponsive to NO
and prone to degradation. Whereas high-affinity ligands for the unoccupied heme pocket of sGC such as
zinc–protoporphyrin IX and the novel NO-independent sGC activator 4-[((4-carboxybutyl){2-[(4-pheneth
ylbenzyl)oxy]phenethyl}amino) methyl [benzoic]acid (BAY 58-2667) stabilized the enzyme, only the latter
activated the NO-insensitive sGC variant. Importantly, in isolated cells, in blood vessels, and in vivo, BAY
58-2667 was more effective and potentiated under pathophysiological and oxidative stress conditions. This
therapeutic principle preferentially dilates diseased versus normal blood vessels and may have far-reaching
implications for the currently investigated clinical use of BAY 58-2667 as a unique diagnostic tool and highly
innovative vascular therapy.
Introduction
Oxidative stress, a risk factor of several cardiovascular disor-
ders, interferes with NO/cyclic GMP (NO/cGMP) signaling
through scavenging of NO and formation of the strong inter-
mediate oxidant peroxynitrite (ONOO–; refs. 1–4). Under these
conditions, endothelial and vascular dysfunction develops, cul-
minating in coronary heart disease, myocardial infarction, and
stroke (5). Substituting NO with organic nitrates that release
NO (NO donors) has been an important principle in cardiovas-
cular therapy for more than a century. However, development
of nitrate tolerance limits their continuous clinical application
and, under oxidative stress and increased formation of ONOO–,
foils the desired therapeutic effect (5). In addition, cardiovas-
cular disease states are accompanied by downregulation of the
activity and expression of the heme-containing NO receptor
soluble guanylyl cyclase (sGC; refs. 6–8). In vitro, the activity
of the NO-sensitive sGC can be directly impaired by ONOO–
(9, 10). Thus ROS both scavenge NO and, via ONOO–, inacti-
vate its receptor with respect to NO sensing. One of the het-
erodimeric enzyme’s redox-sensitive sites is a prosthetic heme
group (11) bound to the smaller (70-kDa) b subunit; oxidation
of its ferrous heme inhibits the NO-mediated activation of sGC
(12). Possible further implications of heme oxidation in regulat-
ing enzyme stability and protein levels are not yet known; even
whether oxidized sGC is present under (patho)physiological
conditions is a matter of debate.
It would therefore be desirable to directly measure the
intracellular molar ratio of Fe2+/Fe3+-containing and heme-free
sGC, specifically against the background of all other cellular
heme proteins with sufficient sensitivity and independent of arti-
ficially changing this ratio by the extraction procedure (see Sup-
plemental Discussion; supplemental material available online
with this article; doi:10.1172/JCI28371DS1). Such methods are
not available to date. In lieu thereof, a recently discovered NO-
and heme-independent activator of sGC, 4-[((4-carboxybutyl)
Nonstandard abbreviations used: BAY 41-2272, 5-cyclopropyl-2-[1-(2-fluoro-ben-
zyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine; BAY 58-2667, 4-[((4-car-
boxybutyl){2-[(4-phenethylbenzyl)oxy] phenethyl}amino) methyl [benzoic]acid; BNP,
B-type natriuretic peptide; cGMP, cyclic GMP; DEA/NO, 2-(N,N-diethylamino)-diaze-
nolate-2-oxide; EC50, 50% effective concentration; GTN, glycerol trinitrate; L-NAME,
N-nitro-L-arginine methylester; NAC, N-acetyl-cysteine; NZW, New Zealand (rabbits);
ODQ, 1H-[1,2,4]oxadiazolo [3,4-a]quinoxalin-1-one; ONOO–, peroxynitrite; PDE,
phosphodiesterase; PE, phenylephrine; PPIX, protoporphyrin IX; SIN-1, 3-morpho-
lino-sydnonimine hydrochloride; sGC, soluble guanylyl cyclase; SHR, spontaneously
hypertensive rats; TG(mRen2)27 rats, hypertensive transgenic renin rats; U46619,
9,11-dideoxy-9a,11a-methanoepoxyprostaglandin F2a; WHHL, Watanabe heritable
hyperlipidemic (rabbits).
Conflict of interest: Johannes-Peter Stasch and Andreas Knorr are currently full-time
employees of Bayer HealthCare. Peter M. Schmidt was employed from 2000 to 2003
by Bayer HealthCare. Harald Lapp is the principal investigator of a clinical study of
patients with acute decompensated heart failure using BAY 58-2667. Adrian Terstee-
gen is currently employed by Bayer HealthCare. Harald H.H.W. Schmidt is coinventor
of a patent on the use of human sGC, owned by vasopharm GmbH, and holds shares
in that company.
Citation for this article: J. Clin. Invest. 116:2552–2561 (2006). doi:10.1172/JCI28371.
Related Commentary, page 2330

research article
The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 9 September 2006 2553
{2-[(4-phenethyl-benzyl)oxy] phenethyl}amino)methyl[benzoic]
acid (BAY 58-2667) appears to be the first experimental tool to
assay the intracellular sGC redox state (13).
Stasch and coworkers have characterized BAY 58-2667 as an
NO-independent sGC activator with unique pharmacological
and biochemical properties such as potent in vitro and in vivo
vasorelaxation even under conditions of nitrate tolerance and
antiaggregatory activity not shared by nitrate-based sGC activa-
tors (13). Furthermore, BAY 58-2667 exhibited an unexpected
biochemical feature, namely the activation of the oxidized/
heme-free state of sGC, indicating that this compound might
be a valuable tool to access this “assumed-to-be” artificial redox
state of sGC.
However, this initial work left major essential clinically and ther-
apeutically relevant points open: Is oxidized/heme-free sGC just
a biochemical artifact, or is it present under physiological condi-
tions as part of a previously unrecognized sGC redox equilibrium?
Are the effects of BAY 58-2667 due to the activation of oxidized
or reduced sGC? Can this putative selectivity be exploited thera-
peutically in diseased versus normal blood vessels, and what are
the long-term consequences of BAY 58-2667 in therapeutically
relevant animal models? And, most important, is the compound
active in normal and diseased human vessels?
In the present work we therefore aimed to go beyond the initial
characterization of BAY 58-2667 and to address these clinically rel-
evant questions. We demonstrate for the first time to our knowledge
that BAY 58-2667 selectively activates a state of sGC that is indistin-
guishable from the NO-insensitive oxidized/heme-free state of the
enzyme, introducing to vascular medicine what we believe to be a
novel mode of sGC activation and vasodilatation. We demonstrate
that ROS such as ONOO–, a hallmark of various cardiovascular dis-
eases, are capable of shifting the sGC redox equilibrium to the ferric
NO-insensitive state. BAY 58-2667 could represent the first thera-
peutic principle that preferentially targets diseased blood vessels,
which we have shown here in vitro and in vivo including in human
vascular disease models, known to be accompanied by increased lev-
els of vascular oxidative stress. Collectively, we hereby extend the ini-
tial discovery and observations of BAY 58-2667 to what we believe to
be a new concept of vascular dysfunction and suggest a therapeutic
approach to this via an enzyme- and redox-specific drug.
Figure 1
Chemical structure of BAY 58-2667.
Figure 2
Effects of competitive porphyrins and the sGC Fe2+/Fe3+ redox ratio
on BAY 58-2667 binding to and activation of sGC. (A) Human sGC
activity in the presence of BAY 58-2667 (filled squares), PPIX (filled
circles), 10 mM BAY 58-2667 and various concentrations of PPIX (open
squares), and 10 mM PPIX and various concentrations of BAY 58-2667
(open circles). Basal activity was 17 ± 3 nmol/mg/min. (B) Heme-
free rat sGC activity in the presence of BAY 58-2667 (filled squares),
PPIX (filled triangles), and 1 mM BAY 58-2667 and various concentra-
tions of PPIX (open circles). Basal activity was 90 ± 7 nmol/mg/min.
(C) Zn-PPIX (open squares), Fe2+-PPIX (filled circles), and Mn-PPIX
(open circles) completely inhibited activation by 1 mM BAY 58-2667
(filled squares) of heme-free rat sGC. (D) Zn-PPIX displaced 3H–BAY
58-2667 (100 nM) from heme-free rat sGC. (E) Rat sGC activity
in the presence of DEA/NO (filled triangles), BAY 58-2667 (open
squares), BAY 41-2272 (filled circles), and BAY 41-2272 combined
with 10 nM DEA/NO (open circles). (F) Rat sGC activity in the pres-
ence of 100 nM BAY 58-2667 (filled circles) combined with increas-
ing concentrations of ODQ. Open triangles represent controls. (G)
Binding of 100 nM 3H–BAY 58-2667 to sGC in the presence of
increasing concentrations of ODQ. (H–J) Same conditions as E–G
but with heme-free rat sGC. Data are means ± SEM of 3–6 indepen-
dent experiments performed in triplicate.

research article
2554 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 9 September 2006
Results
To establish BAY 58-2667 (Figure 1) as an adequate tool to follow
changes in the intracellular sGC redox equilibrium, we first aimed
to clarify in vitro whether oxidized rather than reduced sGC is the
primary target of sGC activators that are known to bind to the sGC
heme pocket, e.g., BAY 58-2667 and protoporphyrin IX (PPIX).
The majority of a standard recombinant sGC preparation will
be purified in a reduced heme-containing state; however, like for
any other purified heme protein, small amounts of oxidized and
heme-free enzyme will also be present in virtually unpredictable
ratios. Stimulation of such a preparation with the NO donor
2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO; 300 mM)
and PPIX (10 mM) produced a 23- and 32-fold stimulation of
cGMP formation compared with basal enzymatic activity, respec-
tively (data not shown). Similar to PPIX, BAY 58-2667 activated
sGC in a concentration-dependent manner from 0.1 nM to 10 mM
(Figure 2A); however, BAY 58-2667 was more potent and effec-
tive than PPIX (50% effective concentration [EC50], BAY 58-2667,
6.4 ± 0.68 nM; versus PPIX, 18.7 ± 5.24 nM; P < 0.05). When both com-
pounds were coincubated, a maximally activating concentration
of PPIX prevented further activation by BAY 58-2667 (Figure 2A).
Similar results were obtained with a preparation of exclusively
heme-free recombinant sGC (Figure 2B), indicating that BAY
58-2667 and PPIX compete for the same subpopulation and bind-
ing site of heme-free sGC within this enzyme preparation. This
view was further supported by the observed competition between
BAY 58-2667 and non–sGC-activating metalloporphyrins: BAY
58-2667–stimulated sGC activity was blocked by Zn-, Mn-, and
Fe2+-protoporphyrin in a competitive manner (Figure 2C). More-
over, in a radioligand binding study, sGC-bound, 3H-labeled BAY
58-2667 was completely replaced by Zn-PPIX (Figure 2D; IC50,
20 ± 1.3 nM). This result strongly suggests that BAY 58-2667 and
PPIX activate heme-free sGC by binding to the unoccupied heme
pocket. Pharmacologically, PPIX behaved as a partial agonist, BAY
58-2667 as a full agonist, and Zn-PPIX as a competitive antago-
nist at the sGC heme pocket.
In addition to its effect on the heme-free sGC, BAY 58-2667
also activated the heme-containing, oxidized enzyme. To
address the effect of the Fe2+/Fe3+ sGC redox ratio on the BAY
58-2667 response, heme-containing and heme-free sGC were
incubated with increasing concentrations of 1H-[1,2,4]oxa-
diazolo [3,4-a]quinoxalin-1-one (ODQ) in the presence of
BAY 58-2667. Upon exposure to DEA/NO, BAY 58-2667, and
5-cyclopropyl-2-[1-(2-f luoro-benzyl)-1H-pyrazolo[3,4-
b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), native sGC
showed the activation profile of a heme-containing enzyme
(Figure 2E). In the presence of a maximally activating concentra-
tion of 100 nM BAY 58-2667, sGC activity was further enhanced
by ODQ (EC50, ~190 nM; Figure 2F). This concentration-depen-
dent effect of ODQ was consistent with binding studies showing
an ODQ-dependent increase in 3H–BAY 58-2667 binding with a
similar EC50 (~170 nM; Figure 2G). The excellent correlation of
these findings supports the notion that ODQ or other oxidiz-
ing mechanisms shift the redox equilibrium to oxidized, BAY
58-2667–sensitive form. A mechanism by which BAY 58-2667
discriminates between both sGC redox states was suggested
earlier: BAY 58-2667, although a nonporphyrinic structure, is
able to mimic the spatial structure of the heme, allowing direct
competition of both ligands for the sGC heme-binding pocket
(14). However, only the weakly bound oxidized heme (15) can
be effectively replaced by BAY 58-2667. In contrast, no com-
petition with the reduced heme was observed, even at concen-
trations at the limit of solubility of BAY 58-2667, foiling any
classical receptor-binding approach (16). Consistently, at the
heme-free enzyme BAY 58-2667–induced sGC activation and
binding quickly reached saturation that was not influenced by
further addition of ODQ (Figure 2, I and J). DEA/NO alone or
combined with the NO sensitizer BAY 41-2272 showed no sGC
activation (Figure 2H). These data establish in vitro that BAY
58-2667 activates only the heme-deficient enzyme via a direct
interaction with the unoccupied heme-binding pocket or by
displacing the weakly bound, oxidized heme.
Figure 3
Effects of oxidative agents on BAY 58-2667–induced
cGMP accumulation in native cells. (A) Basal (white
bars) and BAY 58-2667–stimulated (black bars)
cGMP accumulation were measured in untreated
(control) porcine pulmonary artery endothelial cells
and cells treated for 30 minutes with 20 mM ODQ. (B)
Basal and BAY 58-2667–stimulated cGMP accumu-
lation was measured in untreated (white bars) human
platelets and human platelets treated with ODQ for
30 minutes (black bars). Basal cGMP level was
0.15 ± 0.01 pmol/106 cells. (C) Basal, 250 mM DEA/NO–
stimulated, and 10 mM BAY 58-2667–stimulated cGMP
accumulation was measured in untreated (white bars)
endothelial cells and cells treated with 500 mM SIN-1
for 24 hours (black bars). Basal cGMP level was
0.16 ± 0.04 pmol/106 cells. Data are means ± SEM
of 3 independent experiments performed in triplicate.
(D) cGMP content of aortic rings of Wistar rats and SHR
(5–6 months of age) exposed to vehicle (white bars),
10 mM BAY 58-2667 (gray bars), or 10 mM BAY 58-2667
plus 10 mM ODQ (black bars). cGMP levels are
expressed as mean ± SEM of 5–10 animals. *P < 0.05;
**P < 0.01; ***P < 0.001.

research article
The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 9 September 2006 2555
Next we addressed whether a BAY 58-2667–sensitive sGC
is present in native endothelial cells. Exposure of endothelial
cells to BAY 58-2667 (10 mM, 3 minutes) produced a small but
significant 3.4-fold increase in cGMP levels that was increased
to 10-fold upon pretreatment with ODQ (20 mM, 30 minutes;
Figure 3A). Similar results were obtained in human platelets
(Figure 3B). BAY 58-2667 had virtually no effect on a broad
range of cyclic nucleotide–metabolizing enzymes (phosphodies-
terase [PDE] inhibition by 10 mM BAY 58-2667 for PDE1, 1%; for
PDE2, 5%; for PDE3, 4%; for PDE4, 2%; for PDE5, 1%; for PDE6,
15%; for PDE7, 2%; for PDE8, –4%; for PDE9, 0%; for PDE11, 4%),
indicating that increased cGMP accumulation must be due to
an effect on cGMP synthesis rather than cGMP degradation. To
investigate whether the effectiveness of BAY 58-2667 to stimu-
late oxidized sGC in intact cells holds true for models of oxida-
tive stress that resemble more vascular disease conditions, we
Figure 4
Effects of BAY 58-2667 on sGC protein levels under normal and oxidative conditions. (A) Endothelial cells were incubated for 24 hours with 10 mM
ODQ, 10 mM BAY 58-2667, or the combination of both compounds. (B) Endothelial cells were treated with different concentrations of BAY 58-2667
for 24 hours. (C) Endothelial cells were incubated for 24 hours with 10 mM methylene blue (MB), 10 mM BAY 58-2667, or the combination of
both compounds. (D) Porcine smooth muscle cells were incubated for 24 hours with 10 mM ODQ, 10 mM BAY 58-2667, or the combination of
both compounds. (E) Endothelial cells were incubated for 24 hours with 500 mM SIN-1, 10 mM BAY 58-2667, or the combination of both com-
pounds. (F) Endothelial cells were treated with different concentrations of Zn-PPIX for 48 hours. (G) sGC a and (H) sGC b protein expression
in endothelial cells preincubated with 10 mM ODQ, 10 mg/ml cycloheximide, or 10 mg/ml emetine for 0, 2, 4, and 8 hours. (I) Endothelial cells
were treated for 24 hours with 250 mM 8-Br-cGMP, 100 mM YC-1, 200 mM of the PDE inhibitor Zaprinast, and 100 mM DEA/NO. All sGC protein
levels were determined by Western blot. Data are expressed as percent of control (means ± SEM of 3–6 independent experiments performed
in triplicate). *P < 0.05; **P < 0.01. On A, C, D, and E, representative blots are shown.

research article
2556 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 9 September 2006
used the ONOO– donor 3-morpholino-sydnonimine hydrochlo-
ride (SIN-1; ref. 17). ONOO–, generated by the reaction between
superoxide and NO, was shown to decrease NO-stimulated sGC
activity (9, 10), and heme oxidation was suggested to be a pos-
sible mechanism for this effect (7). Indeed, when endothelial
cells were treated with 500 mM SIN-1 for 24 hours, a signifi-
cant decrease in NO donor–stimulated cGMP accumulation was
observed while the BAY 58-2667 response increased (Figure 3C).
In agreement with the above findings obtained with the puri-
fied enzyme (Figure 2, C and D), Zn-PPIX (10 mM) selectively
abolished the BAY 58-2667–induced increase in cellular cGMP
while the NO response remained unaffected (data not shown).
Together, these data demonstrate under physiological and more
so under oxidative stress the intracellular occurrence of a BAY
58-2667–sensitive sGC that is indistinguishable from the iso-
lated heme-free and/or oxidized enzyme.
Besides these acute effects of BAY 58-2667 on sGC activ-
ity, we observed a second chronic effect: prolonged exposure
of endothelial cells to BAY 58-2667 resulted in an additional
increase in the protein levels of the heme-binding sGC b1 sub-
unit (Figure 4B), while NO donors, NO sensitizers of sGC like
YC-1, or cGMP analogs had no significant effect — or the oppo-
site effect — on sGC protein levels (Figure 4I). We hypothesize
that this effect of BAY 58-2667 is based on reduced degradation
due to stabilization of the sGC b subunit upon BAY 58-2667
binding to its heme pocket. While the importance of sGC heme
and its redox state for the regulation of enzyme activity is well
understood, its role in regulating sGC stability and protein levels
is unclear. Conflicting data regarding the effects of oxidation on
sGC protein levels have been reported. ODQ prevents the NO-
induced decrease of sGC protein levels in smooth muscle cells
(18), whereas treatment of chromaffin cells with ODQ results in a
loss of sGC protein (19). In good agreement with these latter data,
we found a decrease in sGC protein levels upon exposing por-
cine endothelial (Figure 4A) or smooth muscle cells (Figure 4D)
to ODQ. Mechanistically, the effect of ODQ on sGC protein is
most likely due to ubiquitin-dependent protein degradation
(Supplemental Figures 2 and 3). The ODQ-induced decrease
in sGC protein was faster than by inhibiting protein synthesis
(cycloheximide or emetine; Figure 4, G and H). Another sub-
stance known to oxidize sGC heme, methylene blue (20–22),
also decreased sGC protein levels (Figure 4C). Importantly, the
effects of all tested heme oxidants — ODQ, SIN-1 (Figure 4E),
and methylene blue — on sGC protein levels were completely
prevented in the presence of BAY 58-2667. These data suggest
that oxidation of sGC heme results not only in the loss of NO-
sensitive sGC activity but also in ubiquitin-dependent sGC pro-
tein degradation (Supplemental Figures 2 and 3).
The latter mechanism apparently involves a reduced affinity of
the apo-enzyme for the oxidized heme and possible loss of the pros-
thetic heme group (14), which is why heme mimetics that occupy
the heme binding pocket, stabilize the enzyme and consequently
increase steady-state levels of sGC protein. To determine whether
the stabilization of sGC is a unique feature of BAY 58-2667 or
whether it is a general property of ligands to the sGC b1 heme-
binding pocket, we examined the effects of Zn-PPIX, which has
high affinity for sGC (Kd, 16 nM; ref. 23). Zn-PPIX also increased
sGC b1 protein levels without affecting sGC a1 levels, similar to the
effects of BAY 58-2667 (Figure 4F).
While other interpretations of these data cannot be excluded,
a likely explanation is that occupation of the heme-binding site
with high-affinity heme mimetics in general prevents oxida-
tion-induced, ubiquitin-dependent sGC degradation. The sGC
a subunit is unable to bind heme, making this subunit insensi-
tive to heme oxidants but also to stabilizing heme-site ligands.
The a subunit is, however, affected indirectly by the oxidation-
induced decrease of the b protein levels, as sGC is only stable as
a heterodimer and single sGC subunits are prone to degrada-
tion (24). This explains why the protein levels of both subunits
decreased in parallel upon addition of different heme oxidants.
Once high-affinity heme-site ligands such as BAY 58-2667 or
Zn-PPIX were added, the sGC b subunit was stabilized, result-
ing in the observed strong increase in this protein’s expression.
The availability of the stabilized b subunit conserves the sGC
Figure 5
Effects of ONOO– or the ONOO– donor, SIN-1,
on relaxation of rat thoracic aorta. Vessels were
incubated in with and without ONOO– (1 mM
lumazine, 58 m/l XO, 200 mM SPER-NO) and/or
1–10 mM NAC for 1.5 hours or 100 mM SIN-1
for 24 hours. Thereafter, relaxation in responses
to (A) acetylcholine (ACh), (B) DEA/NO, or (C
and D) BAY 58-2667 were measured. (C) 1 mM
(filled diamonds) and 10 mM (open triangles)
NAC, ONOO– (filled circles), and 10 mM NAC
plus ONOO– (open circles) were compared to a
1.5-hour sham incubation (open squares). Data
are presented as means ± SEM of 4 (acetylcho-
line), 6 (DEA/NO), or 9 (BAY 58-2667) indepen-
dent experiments performed in duplicate.

research article
The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 9 September 2006 2557
a protein on the level of controls as stable heterodimers can be
formed. However, as the a subunit cannot bind heme or other
heme-site ligands, its physiological turnover is not affected,
explaining the lack of increased a protein levels as observed for
the b subunit. These data indicate that under oxidizing condi-
tions, BAY 58-2667 and Zn-PPIX preserve stable heterodimeric
sGC levels in an active (BAY 58-2667) or inactive (Zn-PPIX) state
and, in addition, result in the accumulation of nonfunctional b
mono- or probably homodimers (25).
Moreover, our data indicate that BAY 58-2667 may represent
a unique vasodilator that is of particular use under conditions
of oxidative stress. Therefore, we extended our experiments
to functionally analyze isolated blood vessels. Rat aortic ring
preparations were incubated with and without the endogenous
oxidant ONOO– for 1.5 hours or with SIN-1 for 24 hours, and
the relaxation of phenylephrine (PE) precontraction was mea-
sured in response to acetylcholine, DEA/NO, or BAY 58-2667.
Contractility of the vessels was not affected by the long-term
treatment with SIN-1 (data not shown). However, the maximal
relaxation in response to acetylcholine was impaired (ONOO–,
48.8% ± 6.6%; versus control, 90.3% ± 1.4%; Figure 5A). The NO
donor DEA/NO produced complete relaxation of both control
Figure 6
Vasorelaxing effects of BAY 58-2667 in vitro and in
vivo. (A) BAY 58-2667–induced relaxation of PE pre-
contracted aorta from Wistar rats (circles) and aged
SHR (triangles) with (open symbols) and without
(filled symbols) ODQ. (B) BAY 58-2667– (triangles)
or SNP–induced (squares) inhibition of PE precon-
tracted saphenous arteries from NZW (filled sym-
bols) and WHHL rabbits (open symbols). (C) BAY
58-2667– (triangles) or GTN-induced (circles) inhi-
bition of U46619 precontracted aorta from ApoE–/–
mice on normal (open symbols) or high-fat diet (filled
symbols). (D) BAY 58-2667–induced inhibition of
PE precontracted human mesocolon arteries from
patients with (open circles) and without (filled circles)
type 2 diabetes. Means ± SEM of 6–12 vessels shown
in A–D. (E) Effect of i.v. BAY 58-2667 (10 mg/kg)
with (gray triangles) and without (filled triangles)
ODQ pretreatment (2 mg/kg i.v., 10 minutes before
BAY 58-2667) or vehicle (open circle) on MAP in
anesthetized rats (n = 4). Baseline was 117 ± 3
to 128 ± 4 mmHg. (F) Effect of oral BAY 58-2667
(filled circles, 3.0 mg/kg) and vehicle (open circles)
on MAP in conscious Wistar rats. Baseline was
106 ± 4 and 102 ± 3 mmHg (control versus treated,
respectively; n = 6). (G) Effect of oral BAY 58-2667
(filled circles, 3.0 mg/kg) and vehicle (open circles)
on MAP in conscious SHR. Baseline was 135 ± 11
and 133 ± 4 mmHg (control versus treated, respec-
tively; n = 6). In E–G, predrug values of each group
were normalized to 100%. (H) Survival rate in BAY
58-2667–treated (filled circles, 3 mg/kg orally twice
daily) and untreated (open triangles) TGR(mRen2)27
under l-NAME. (I) Plasma levels of BNP, renin, cre-
atinine, and urea in BAY 58-2667–treated (black
bars) versus control animals (white bars). Values
are means ± SEM. ***P < 0.001.

research article
2558 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 9 September 2006
and ONOO–-treated vessels, but its potency was decreased 10-
fold after treatment with ONOO– (EC50, ONOO–, 6.20 ± 0.24
logM; versus control, 7.34 ± 0.04 log M; Figure 5B). In contrast,
the sensitivity to BAY 58-2667 was increased after treatment
with both ONOO– (EC50, ONOO–, 9.34 ± 0.11 logM; versus
control, 8.27 ± 0.05 logM; Figure 5C) and SIN-1 (EC50, SIN-1,
11.35 ± 0.06 logM; versus control, 10.64 ± 0.05 logM, Figure 5D).
Moreover, following 1.5 hours of treatment with ONOO–
(Figure 5C) and 24 hours of treatment with SIN-1 (Figure 5D), the
increase in the maximum response to BAY 58-2667 was striking.
Consistently, pretreatment with the reductant N-acetyl-cysteine
(NAC) dramatically lowered the effectiveness of BAY 58-2667
(Figure 5C). Thus the vasorelaxant potency and efficacy of BAY
58-2667 is increased under conditions of oxidative stress.
To validate this unprecedented feature of a vasodilator drug,
we investigated vascular reactivity in 4 distinct disease models
involving endogenous oxidative stress rather than exogenously
induced oxidative stress: aged spontaneously hypertensive rats
(SHR), Watanabe heritable hyperlipidemic (WHHL) rabbits,
ApoE–/– mice on high-fat diet, and human mesocolon arteries
from patients with type 2 diabetes.
BAY 58-2667 inhibited the PE-induced contraction of aortic rings
from normal and aged SHR in a concentration-dependent man-
ner (IC50, normal SHR, 1.23 ± 0.16 nM; aged SHR, 0.35 ± 0.14 nM;
P < 0.01), and the addition of ODQ (10 mM) shifted the concentra-
tion-response curves leftward (IC50, normal SHR, 0.15 ± 0.03 nM;
aged SHR, 0.09 ± 0.013 nM; P < 0.01; Figure 6A). These results
show that BAY 58-2667 more potently relaxes vessels from hyper-
tensive rats than normotensive rats if the heme group of sGC
is oxidized. To validate that this enhanced vasorelaxation was
indeed mediated by sGC stimulation, we measured the effects of
BAY 58-2667 on vascular cGMP levels in aortas taken from nor-
mal rats and SHR with and without 10 mM ODQ pretreatment.
Figure 3D shows that exposure of 10 mM BAY 58-2667 induced
significantly higher cGMP levels in aortas from hypertensive ver-
sus normotensive rats and that this cGMP increase was further
potentiated in the presence of ODQ.
In WHHL rabbits a marked attenuation of the endothelium-
dependent vasodilatation has been identified as a characteris-
tic of early stages of atherosclerosis related to enhanced inac-
tivation of endothelium-derived NO by superoxide (26). BAY
58-2667 inhibited PE-induced contractions of saphenous artery
rings from WHHL rabbits more potently than of control New
Zealand (NZW) rabbits (IC50, WHHL, 0.1 ± 0.012 nM; NZW,
1.2 ± 0.09 nM; P < 0.001). However, the NO donor sodium nitro-
prusside relaxed precontracted arteries from WHHL and NZW
rabbits with no significant difference (IC50, NZW, 105 ± 24 nM;
WHHL, 153 ± 25 nM; Figure 6B).
To confirm these data in a third animal model for vascular dis-
ease, we investigated the vasorelaxing effects of BAY 58-2667 and
the NO donor glycerol trinitrate (GTN) on precontracted aortas
from ApoE–/– mice fed standard and high-fat diets. BAY 58-2667
inhibited precontracted vessels (IC50, standard diet, 4.0 ± 0.28 nM;
high-fat diet, 0.7 ± 0.09 nM; P < 0.001; Figure 6C). In contrast,
the relaxation response to GTN was impaired in ApoE–/– mice on
the high-fat diet (IC50, standard diet, 67.5 ± 20.8 nM; high-fat diet,
119 ± 54.3 nM; P < 0.01). These data suggest that a milder degree
of sGC oxidation occurs physiologically and that accumulated oxi-
dized sGC under pathophysiological conditions represents a pool
that can be selectively stimulated by BAY 58-2667 to cause selective
vasodilatation of pathological blood vessels.
We then sought to apply these observations to human tissue.
Since diminished activity of vascular sGC has also been reported
in animal models of type 2 diabetes (27), we investigated the relax-
ing effect of BAY 58-2667 in human arteries taken from human
mesocolon specimens obtained during bypass surgery from
patients with and without type 2 diabetes. Indeed, BAY 58-2667
relaxed arteries from diabetic patients more potently than controls
(IC50, 0.03 ± 0.002 nM; versus control, 0.39 ± 0.02 nM; P < 0.001;
Figure 6D). Thus selective targeting of heme-oxidized sGC extends
to human forms of oxidative stress and vascular disease.
Finally, we wanted to investigate the effectiveness and selectivity
of BAY 58-2667 at the in vivo level with and without ODQ pretreat-
ment. In anesthetized rats, BAY 58-2667 produced a dose-depen-
dent and long-lasting hypotension (Figure 6E). After i.v. admin-
istration of 10 mg/kg BAY 58-2667, a maximal effect of 29 mmHg
was observed. Pretreatment with ODQ induced not only a signifi-
cant increase in effectiveness to 71 mmHg, but it also prolonged the
effect’s duration (Figure 6E). Upon oral administration of 3 mg/kg
BAY 58-2667, the hypotension lasted longer in conscious SHR
than in normotensive Wistar rats (Figure 6, F and G). Given that
chronic inhibition of NO synthesis in rats increases aortic super-
oxide anion production via an activated renin angiotensin sys-
tem (28), the cardiovascular consequences of sGC activation were
evaluated by the long-term effects of BAY 58-2667 in hyperten-
sive transgenic renin rats [TG(mRen2)27 rats] treated with the
NOS inhibitor N-nitro-L-arginine methylester (L-NAME). Systolic
blood pressure, as measured by the tail cuff method, increased
in 11-week-old TG(mRen2)27 rats that received L-NAME from
Figure 7
NO receptor sGC exists in a physiological equilibrium between 2 redox
states, the reduced and NO-sensitive versus the oxidized and heme
mimetic–sensitive form. Under pathophysiological conditions, this rela-
tive ratio is shifted toward the oxidized form, which is selectively tar-
geted by sGC activators such as BAY 58-2667 to overcome the patho-
physiology of impaired NO-cGMP signaling. In addition, high-affinity
heme-site ligands such as Zn-PPIX (Zn) or BAY 58-2667 (BAY) are
able to block the oxidation-accelerated degradation of the sGC a/b
heterodimer, thereby stabilizing and conserving the enzyme protein
levels in an inactive (Zn-PPIX–bound) or active (BAY 58-2667–bound)
form even under oxidizing conditions. Furthermore, heme-site ligands
decelerate the physiological turnover of the sGC b subunit, resulting in
an accumulation of this monomeric protein.

research article
The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 9 September 2006 2559
159 ± 3 mmHg to 176 ± 3 mmHg, whereas in animals treated with BAY
58-2667 the systolic pressure decreased slightly to 150 ± 3 mmHg
(Figure 6H). At the end of the study, plasma B-type natriuretic
peptide (BNP), creatinine, urea, and renin activity were signifi-
cantly lower, reflecting cardiorenal protection effect (Figure 6I).
Most notably, the overall beneficial effect of BAY 58-2667 was
reflected in a significant reduction in mortality. During the course
of study, 9 of 20 control animals died, whereas in the treatment
group all 15 animals stayed alive.
Discussion
Murad, Furchgott, and Ignarro originally outlined the endog-
enous NO-cGMP signaling pathway in blood vessels. Since then,
different ligands and stimulators ranging from small molecu-
lar compounds to proteins have also been shown to stimulate
heme-containing sGC in the cardiovascular system. In 2002, we
described a member of a novel structural class of compounds
(BAY 58-2667) that, in contrast to the NO-independent but
heme-dependent sGC stimulators such as YC-1, BAY 41-2272,
and BAY 41-8543, could activate sGC in its NO-insensitive,
oxidized (or heme-deficient) state (13). The activation of this
presumably artificial state of sGC was an unexpected biochemi-
cal feature and not fully recognized as a drug target. Here we
provide compelling evidence that an sGC that is indistinguish-
able from oxidized/heme-free sGC occurs physiologically and
can be selectively targeted by BAY 58-2667. Most important, the
levels of oxidized/heme-free sGC appear to increase in various
pathophysiological animal models and in human cardiovascular
diseases that are accompanied by increased production of ROS,
suggesting a causal link among vascular dysfunction, ROS for-
mation, heme oxidation, and ultimately heme loss. The selectiv-
ity of BAY 58-2667 for oxidized/heme-free sGC thus allows for
targeting of diseased blood vessels in an unprecedented manner
and with potency in the low nM range.
In order to validate BAY 58-2667 as a potent tool for identifying
oxidized/heme-free sGC in vitro and in vivo, we established BAY
58-2667 as a full agonist at the heme-binding site of sGC, PPIX as
a partial agonist, and Zn-PPIX as a full antagonist. The fact that
Zn-PPIX lowers cGMP levels in vivo suggests that endogenous
ligands for heme-free sGC exist.
Here we describe what we believe to be a new signaling path-
way prevalent under disease conditions. Moreover, we observed
that oxidized sGC seems to be prone to accelerated degradation
through ubiquitinilation. BAY 58-2667 stabilizes heme-free sGC
in a positive feedback loop. This selective vasodilation may in the
future also be used as a functional diagnostic tool in humans.
Potential therapeutic indications may include heart failure, hyper-
tension, pulmonary hypertension, renal failure, and peripheral
arterial occlusive disease.
In summary, our data indicate that sGC exists under physiologi-
cal conditions in an equilibrium between its reduced and oxidized
state; oxidative stress shifts this ratio more to the NO-insensitive
ferric/heme-free form (Figure 7). The selectivity of BAY 58-2667
to activate this oxidation-impaired/heme-free form of sGC has
important implications for the pharmacology of sGC, the funda-
mental understanding of sGC regulation in NO signaling, and the
potential use of BAY 58-2667 as a diagnostic tool and as a highly
innovative therapy for vascular disease; for the first time to our
knowledge, selective vasodilatation of diseased blood vessels under
oxidative stress is possible.
Methods
BAY 58-2667 was synthesized as described previously (29) at a purity of
98.6% as determined by HPLC, NMR, and liquid chromatography mass
spectroscopy. The compound may be obtained from Bayer HealthCare via
a material transfer agreement. BAY 41-2272, DEA/NO, and ODQ were pur-
chased from Alexis Biochemicals; Axxora. All other chemicals of analytical
grade were obtained from Sigma-Aldrich.
Purification of sGC and activity assay. Recombinant human and rat sGC
was expressed using the Sf9/baculovirus system and purified as described
previously (3, 25, 30). Measurement of human recombinant sGC activity
with subsequent determination of cGMP content by enzyme immunoas-
say was performed as described previously (31). Activity of recombinant
rat sGC was measured by formation of [32P]-cGMP from [a-32P]-GTP (30).
Removal of the sGC heme moiety and subsequent removal of the detergent
was performed as described previously (30).
Cell culture. Porcine pulmonary artery endothelial cell culture was per-
formed as described previously (32). Smooth muscle cells were isolated
enzymatically by incubation of the aortic inner surface with collagenase
type CLS II (0.5 mg/ml, 10 min at room temperature) after removal of
endothelial cells. Growth media used were M199 for endothelial cells
and DMEM (Sigma-Aldrich) for smooth muscle cells, both supple-
mented with 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/ml
streptomycin. Passages 6–8 (for smooth muscle cells) or confluent cell
monolayers from the second passage (for endothelial cells) were used for
experiments. To measure intracellular cGMP content, cells were stimu-
lated with the compounds indicated in Figures 3 and 4, medium was
removed, and 70% ethanol was added. After evaporation of ethanol, cells
were homogenized in assay buffer (BIOTREND), and cGMP was mea-
sured by enzyme immunoassay according to the manufacturer’s instruc-
tions. Alternatively, for Western blot analysis, cells were lysed in 250 ml
Roti-Load (Carl Roth) sample buffer preheated to 95°C and then boiled
for an additional 10 minutes.
Aortic cGMP. Determination of vascular cGMP content of rat aortic rings
was performed as described previously (33).
Human platelets. Isolation of human platelets and cGMP measurements
were performed as described previously (13).
Western blotting. Immunodetection of sGC proteins was performed as
described previously (31). Detection of sGC a1/b1 was performed using
subunit-specific antibodies (31). Quantification of luminescent signals
was performed using Kodak Imager Station 440CF.
Receptor binding study. Binding studies were performed as described
previously (16).
PDE inhibition. The preparation of PDE isoenzymes and inhibition of
PDE enzyme activity have been previously described (34).
Human vessels. We included consecutive patients scheduled for elec-
tive bowel surgery if they fulfilled the following inclusion criteria: aged
50–75 years, no risk factors for atherosclerosis or diabetes or at least 2
risk factors for atherosclerosis and type 2 diabetes of at least 2 years’
duration, and written informed consent. Patients were excluded if 1 or
more of the following criteria were fulfilled: age less than 50 or greater
than 75 years, type 1 diabetes, or vasodilator therapy during surgery.
The medical history of patients eligible for the study was recorded using
a standardized questionnaire that contained the following items: type
of surgery and anesthesia, additional diseases and/or events, standard
laboratory parameters, and regular medication. Vessels with a diameter
of 1.5–2.5 mm were dissected from the mesocolon, and segments of
2.0 cm were gently isolated from fat and loose connective tissue. The
preparations were immediately collected into vials with chilled modi-
fied Krebs Henseleit buffer and immediately transferred for experi-
ments. The study was carried out in adherence to the Declaration of

research article
2560 The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 9 September 2006
Helsinki and approved by the ethics committee of the University of Wit-
ten/Herdecke (no. 82/2004).
Vasorelaxation studies. Rat thoracic aortas and human vessels were rap-
idly isolated, and vessels were prepared as described previously (26, 35).
After isolation, vessels were transferred into Krebs buffer containing
additional 2% fetal calf serum, 100 U/ml penicillin, and 100 mg/ml strep-
tomycin. Vessels were incubated in this solution for 24 hours at 37°C
under an atmosphere of 5% CO2 and saturated humidity. After mount-
ing, vessels were allowed to equilibrate for 90 minutes before addition
of any drugs. Krebs buffer was renewed every 15 minutes throughout
the experimental protocol, except during the generation of concentra-
tion-response curves. After the normalization procedure, vessels were
contracted with a high-potassium solution (80 mM KCl) to determine
whether they were viable. After precontraction of the vessels with PE
(60–80% of maximum) until they reached a stable contraction plateau,
cumulative concentration-dependent relaxation to acetylcholine, DEA/
NO, GTN, or BAY 58-2667 was assessed.
Vasorelaxing measurements on saphenous arteries from rabbits at the
age of about 1.5 years (about 2–3 kg, male NZW rabbits, E.S.D. Romans
Elevage Scientifique des Dombes; male WHHL rabbits, CRP: WHHL
Covance Research Products) were performed as previously described (13).
Male ApoE–/– mice at 6–8 weeks of age were fed a normal diet or a western-
type diet (21% raw fat, 0.15% cholesterol, 19.5 casein; Ssniff) for 16 weeks. Age-
matched C57BL/6J mice fed a normal diet were used as wild-type controls.
Animals were killed by decapitation under general anesthesia and aortas
were isolated (1.5 mm) and suspended immediately under an initial tension
of approximately 0.5 g in 5 ml organ bath containing Krebs-Henseleit solu-
tion (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM
KH2PO4, 25 mM NaHCO3, 5.5 mM D-glucose, and 25 mM EDTA-Ca-salt,
pH 7.4) at 37°C. Contractions were measured isometrically with Statham
UC2 strain gauges connected to a DASI802HC data acquisition board
(Keithley). Rings were precontracted by potassium chloride (100 nM)
3 times. Each contraction was followed by a series of 16 washing cycles and a
resting period of 28 minutes. The aortic rings were subsequently contracted
with the thromboxane analog 9,11-dideoxy-9a,11a-methanoepoxyprosta-
glandin F2a (U46619; maximal concentration, 3 × 10–9 g/ml). When the
contractile response reached a plateau, the test compound was cumula-
tively added to the bath solution. The relaxation responses obtained were
expressed as a percentage of the maximal contraction evoked by U46619.
Hemodynamics in anesthetized rats. Measurements were performed as previ-
ously described (13). Vehicle or ODQ was applied 10 minutes before BAY
58-2667 administration in a volume of 0.1 ml/kg. Both compounds were
administered i.v. in a solution of Transcutol/Cremophor EL/physiological
saline (10:10:80 vol/vol/vol) in a volume of 1 ml/kg.
Twenty-four-hour blood pressure measurements in conscious SHR and Wistar rats.
Measurements were performed as previously described (13).
Long-term in vivo study. Eleven-week-old TGR(mRen2)27 rats were random-
ized in 2 groups, a control group and a group treated with BAY 58-2667.
Rats of both groups were given L-NAME in their drinking water (500 mg/l);
rats of the treatment group additionally received 10 mg/kg BAY 58-2667
orally twice daily for 5 weeks, whereas the control group received no
treatment. The substance was administered as a suspension in Transcu-
tol/Cremophor EL/water (10:20:70 vol/vol/vol) by gavage. Systolic blood
pressure was measured weekly by the tail-cuff method. Blood samples
were taken at the end of the study and collected after decapitation into
chilled tubes containing EDTA. BNP was determined after extraction
using C18-cartridges (Bond Elut; Varian Inc.) with a radioimmunoassay
kit (BIOTREND). Plasma renin activity was determined by measurement
of angiotensin I accumulated in the plasma samples during incubation
for 1 hour at 37°C, pH 6.0, and was measured using a commercial radio-
immunoassay kit (Sorin Biomedia).
Statistics. Data are reported as means ± SEM. Differences were assessed
by 1-way repeated-measures ANOVA followed by Bonferroni test for com-
parison of means. Statistical comparisons of IC50 values were performed
by paired Student’s t test. All statistical calculations were performed using
GraphPad Prism 3.0 software (GraphPad Software).
Acknowledgments
This study was supported by grants from Deutsche Forschungs-
gemeinschaft (SFB547/C7 and C10) and the National Health &
Medical Research Council and the National Heart Foundation,
Australia. P.M. Schmidt is the recipient of an Alexander-von-Hum-
boldt Lynen fellowship.
Received for publication March 1, 2006, and accepted in revised
form July 11, 2006.
Address correspondence to: Harald H.H.W. Schmidt or Peter M.
Schmidt, Monash University, School of Biomedical Sciences,
Department of Pharmacology Wellington Road, Melbourne,
Clayton, Victoria 3800, Australia. Phone: 61-3-9905-5752; Fax:
61-3-9905-5729; E-mail: Harald.Schmidt@med.monash.edu.au
(H.H.H.W. Schmidt). Phone: 61-3-990-20218; Fax: 61-3-990-55851;
E-mail: Peter.Schmidt@med.monash.edu.au (P.M. Schmidt).
1. Hare, J.M. 2004. Nitroso-redox balance in the car-
diovascular system. N. Engl. J. Med. 351:2112–2114.
2. Harrison, D.G. 1997. Endothelial function and oxi-
dant stress. Clin. Cardiol. 20:II-11-17.
3. Melichar, V.O., et al. 2004. Reduced cGMP signal-
ing associated with neointimal proliferation and
vascular dysfunction in late-stage atherosclerosis.
Proc. Natl. Acad. Sci. U. S. A. 101:16671–16676.
4. Pryor, W.A., and Squadrito, G.L. 1995. The chem-
istry of peroxynitrite: a product from the reac-
tion of nitric oxide with superoxide. Am. J. Physiol.
268:L699–L722.
5. Warnholtz, A., et al. 2002. Adverse effects of nitro-
glycerin treatment on endothelial function, vas-
cular nitrotyrosine levels and cGMP-dependent
protein kinase activity in hyperlipidemic Watanabe
rabbits. J. Am. Coll. Cardiol. 40:1356–1363.
6. Bauersachs, J., et al. 1998. Vasodilator dysfunction
in aged spontaneously hypertensive rats: changes
in NO synthase III and soluble guanylyl cyclase
expression, and in superoxide anion production.
Cardiovasc. Res. 37:772–779.
7. Kloss, S., Bouloumie, A., and Mulsch, A. 2000. Aging
and chronic hypertension decrease expression of
rat aortic soluble guanylyl cyclase. Hypertension.
35:43–47.
8. Ruetten, H., Zabel, U., Linz, W., and Schmidt, H.H.
1999. Downregulation of soluble guanylyl cyclase
in young and aging spontaneously hypertensive
rats. Circ. Res. 85:534–541.
9. Mulsch, A., et al. 2001. Effects of in vivo nitroglyc-
erin treatment on activity and expression of the
guanylyl cyclase and cGMP-dependent protein
kinase and their downstream target vasodilator-
stimulated phosphoprotein in aorta. Circulation.
103:2188–2194.
10. Weber, M., Lauer, N., Mulsch, A., and Kojda, G.
2001. The effect of peroxynitrite on the catalytic
activity of soluble guanylyl cyclase. Free Radic. Biol.
Med. 31:1360–1367.
11. Ignarro, L.J., Degnan, J.N., Baricos, W.H., Kadowitz,
P.J., and Wolin, M.S. 1982. Activation of purified
guanylate cyclase by nitric oxide requires heme.
Comparison of heme-deficient, heme-reconstitut-
ed and heme-containing forms of soluble enzyme
from bovine lung. Biochim. Biophys. Acta. 718:49–59.
12. Schrammel, A., Behrends, S., Schmidt, K., Koes-
ling, D., and Mayer, B. 1996. Characterization of
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one as a
heme-site inhibitor of nitric oxide-sensitive guanylyl
cyclase. Mol. Pharmacol. 50:1–5.
13. Stasch, J.P., et al. 2002. NO- and haem-indepen-
dent activation of soluble guanylyl cyclase: molec-
ular basis and cardiovascular implications of a
new pharmacological principle. Br. J. Pharmacol.
136:773–783.
14. Schmidt, P.M., Schramm, M., Schroder, H., Wun-
der, F., and Stasch, J.P. 2004. Identification of resi-
dues crucially involved in the binding of the heme
moiety of soluble guanylate cyclase. J. Biol. Chem.
279:3025–3032.
15. Hobbs, A.J. 2000. Soluble guanylate cyclase [review].
Expert Opin. Ther. Targets. 4:735–749.
16. Schmidt, P., Schramm, M., Schroder, H., and
Stasch, J.P. 2003. Receptor binding assay for nitric
oxide- and heme-independent activators of soluble

research article
The Journal of Clinical Investigation http://www.jci.org Volume 116 Number 9 September 2006 2561
guanylate cyclase. Anal. Biochem. 314:162–165.
17. Darley-Usmar, V.M., Hogg, N., O’Leary, V.J., Wil-
son, M.T., and Moncada, S. 1992. The simultane-
ous generation of superoxide and nitric oxide can
initiate lipid peroxidation in human low density
lipoprotein. Free Radic. Res. Commun. 17:9–20.
18. Filippov, G., Bloch, D.B., and Bloch, K.D. 1997.
Nitric oxide decreases stability of mRNAs encod-
ing soluble guanylate cyclase subunits in rat pul-
monary artery smooth muscle cells. J. Clin. Invest.
100:942–948.
19. Ferrero, R., and Torres, M. 2002. Prolonged expo-
sure of chromaffin cells to nitric oxide down-regu-
lates the activity of soluble guanylyl cyclase and
corresponding mRNA and protein levels. BMC
Biochem. 3:26.
20. Luo, D., Das, S., and Vincent, S.R. 1995. Effects of
methylene blue and LY83583 on neuronal nitric
oxide synthase and NADPH-diaphorase. Eur. J.
Pharmacol. 290:247–251.
21. Mayer, B., Brunner, F., and Schmidt, K. 1993.
Novel actions of methylene blue. Eur. Heart J.
14(Suppl. I):22–26.
22. Mayer, B., Brunner, F., and Schmidt, K. 1993. Inhi-
bition of nitric oxide synthesis by methylene blue.
Biochem. Pharmacol. 45:367–374.
23. Serfass, L., and Burstyn, J.N. 1998. Effect of heme
oxygenase inhibitors on soluble guanylyl cyclase
activity. Arch. Biochem. Biophys. 359:8–16.
24. Mergia, E., Friebe, A., Dangel, O., Russwurm, M.,
and Koesling, D. 2006. Spare guanylyl cyclase NO
receptors ensure high NO sensitivity in the vascular
system. J. Clin. Invest. 116:1731–1737. doi:10.1172/
JCI27657.
25. Zabel, U., Hausler, C., Weeger, M., and Schmidt,
H.H.H.W. 1999. Homodimerization of soluble gua-
nylyl cyclase subunits. Dimerization analysis using
a glutathione s-transferase affinity tag. J. Biol. Chem.
274:18149–18152.
26. Warnholtz, A., et al. 1999. Increased NADH-oxi-
dase-mediated superoxide production in the early
stages of atherosclerosis: evidence for involve-
ment of the renin-angiotensin system. Circulation.
99:2027–2033.
27. Witte, K., et al. 2002. Dysfunction of soluble gua-
nylyl cyclase in aorta and kidney of Goto-Kakizaki
rats: influence of age and diabetic state. Nitric oxide.
6:85–95.
28. Kitamoto, S., et al. 2000. Chronic inhibition of nitric
oxide synthesis in rats increases aortic superoxide
anion production via the action of angiotensin II.
J. Hypertens. 18:1795–1800.
29. Alonso-Alija, C., et al. 2001. Novel derivatives of
dicarboxylic acid having pharmaceutical properties.
World Intellectual Property Organization patent
WO/2001/019780, filed August 31, 2000, and
issued March 22, 2001. http://www.wipo.int/
pctdb/en/wo.jsp?KEY=01/19780.010907.
30. Hoenicka, M., et al. 1999. Purified soluble guanylyl
cyclase expressed in a baculovirus/Sf9 system: stim-
ulation by YC-1, nitric oxide, and carbon monoxide.
J. Mol. Med. 77:14–23.
31. Nedvetsky, P.I., Kleinschnitz, C., and Schmidt,
H.H.H.W. 2002. Regional distribution of protein
and activity of the nitric oxide receptor, soluble
guanylyl cyclase, in rat brain suggests multiple
mechanisms of regulation. Brain Res. 950:148–154.
32. Zabel, U., et al. 2002. Calcium-dependent mem-
brane association sensitizes soluble guanylyl
cyclase to nitric oxide. Nat. Cell Biol. 4:307–311.
33. Priviero, F.B., et al. 2005. Mechanisms underlying
relaxation of rabbit aorta by BAY 41-2272, a nitric
oxide-independent soluble guanylate cyclase acti-
vator. Clin. Exp. Pharmacol. Physiol. 32:728–734.
35. Wunder, F., et al. 2005. Characterization of the first
potent and selective PDE9 inhibitor using a cGMP
reporter cell line. Mol. Pharmacol. 68:1775–1781.
34. Arun, K.H., Kaul, C.L., and Ramarao, P. 2005. AT1
receptors and L-type calcium channels: functional
coupling in supersensitivity to angiotensin II in
diabetic rats. Cardiovasc. Res. 65:374–386.