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Review

This Review is part of a thematic series on Cyclic GMP–Generating Enzymes and Cyclic GMP–Dependent
Signaling, which includes the following articles:

Regulation of Nitric Oxide–Sensitive Guanylyl Cyclase

Cyclic GMP Phosphodiesterases and Regulation of Smooth Muscle Function


Structure, Regulation, and Function of Membrane Guanylyl Cyclase Receptors, With a Focus on GC-A
Cyclic GMP–Dependent Protein Kinases and the Cardiovascular System: Insights From Genetically Modified Mice
Regulation of Gene Expression by Cyclic GMP
Explaining the Phenomenon of Nitrate Tolerance Rudi Busse, Editor

Regulation of Nitric Oxide–Sensitive Guanylyl Cyclase


Andreas Friebe, Doris Koesling

Abstract—In this review, we outline the current knowledge on the regulation of nitric oxide (NO)–sensitive guanylyl
cyclase (GC). Besides NO, the physiological activator that binds to the prosthetic heme group of the enzyme, two novel
classes of GC activators have been identified that may have broad pharmacological implications. YC-1 and YC-1–like
substances act as NO sensitizers, whereas the substance BAY 58-2667 stimulates NO-sensitive GC NO-independently
and preferentially activates the heme-free form of the enzyme. Sensitization and desensitization of NO/cGMP signaling
have been reported to occur on the level of NO-sensitive GC; in the present study, an alternative mechanism is
introduced explaining the adaptation of the NO-induced cGMP response by a long-term activation of the cGMP-
degrading phosphodiesterase 5 (PDE5). Finally, regulation of GC expression and a possible modulation of GC activity
by other factors are discussed. (Circ Res. 2003;93:96-105.)
Key Words: cyclic GMP 䡲 guanylyl cyclase 䡲 nitric oxide 䡲 phosphodiesterase 䡲 sensitization/desensitization

N itric oxide (NO)–sensitive guanylyl cyclase (NO-


sensitive GC) is generally accepted as the most impor-
tant receptor for the signaling molecule NO. This enzyme is
The NO-induced cGMP signal is conveyed intracellularly by
the activation of several effector molecules: cGMP-
dependent protein kinases, cGMP-regulated phosphodiester-
also known as soluble or cytosolic GC, a designation chosen ases, and cGMP-gated ion channels. Most of the cGMP
to distinguish the enzyme from its membrane-bound counter- effects have been shown to be mediated by the cGMP-
part. Because recently one of the isoforms of the “soluble” dependent kinase. Although the occurrence of cGMP-gated
GC has been found to associate with the cell membrane, we channels has been demonstrated outside sensory cells, their
will use the term “NO-sensitive” GC throughout this review. functional role remains to be established. The NO/cGMP
NO-sensitive GC is a heterodimer consisting of two dif- signaling cascade (Figure 1) is of importance in the cardio-
ferent subunits termed ␣ and ␤. The enzyme was shown to vascular and nervous systems, where it controls smooth
contain heme as a prosthetic group.1 Stimulation of the muscle relaxation,2,3 modulation of synaptic transmission,4 – 6
enzyme by its physiological activator NO leads to a tremen- and inhibition of platelet aggregation.7,8
dous, up to 200-fold increase in catalytic rate, ie, the Although cGMP-forming activity was first described in
conversion of GTP to cyclic guanosine monophosphate 1969,9 –13 it took until the mid-1970s to find out that there are
(cGMP). NO-sensitive GC, similar to other nucleotide- two different types of GC,14 which were subsequently found
converting enzymes, requires Mg2⫹ as cofactor for catalysis. to differ not only in their cellular localization (cytosolic

Original received March 6, 2003; revision received June 3, 2003; accepted June 5, 2003.
From Abteilung für Pharmakologie, Medizinische Fakultät, Ruhr-Universität Bochum, Bochum, Germany.
Correspondence to Doris Koesling, Abteilung für Pharmakologie, Medizinische Fakultät, Ruhr-Universität Bochum, Universitätsstr. 150, 44780
Bochum, Germany. E-mail doris.koesling@ruhr-uni-bochum.de
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000082524.34487.31

96
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Friebe and Koesling Regulation of NO-Sensitive Guanylyl Cyclase 97

pyramidal cells, with nNOS being expressed in the postsyn-


aptic side underlining the potential role of NO/cGMP signal-
ing in the regulation of synaptic plasticity.25 Nevertheless, ␣2
is not the only ␣ subunit in brain; ␣1 occurred in similar
amounts. In all other tissues tested, the ␣1 subunit was the
major occurring ␣ subunit.26 It appears that the expression of
␣1 subunit correlates with the degree of vascularization.
Like ␣2, the ␤2 subunit was identified by homology
screening.27 However, existence of the ␤2 subunit on the
protein level appears unlikely; in the human gene, a base
deletion has been described that results in a shift of the
reading frame excluding functional expression of the pro-
tein.28 Quantitative PCR analysis of mouse tissue revealed
virtually no mRNA for the ␤2 subunit.26 In addition, no
convincing evidence of a functionally active ␤2-containing
dimer has been presented so far.
Figure 1. NO/cGMP signaling cascade. NO endogenously pro- NO-sensitive GC is catalytically active only as a het-
duced by NO synthases or released from exogenously applied erodimer.29,30 Homodimeric enzymes (␣1␣1and ␤1␤1) have
NO donors activates NO-sensitive GC and leads to increased
been purified from Sf9 cells; however, no enzymatic activity
synthesis of cGMP. This intracellular messenger in turn modu-
lates the activity of cGMP-dependent kinases, cGMP-gated ion of these homodimers has been detected.31
channels, and cGMP-regulated phosphodiesterases. These A comparison of the primary structure shows that the
effectors are involved in the regulation of several physiological subunits can be divided into three domains: a C-terminal
functions in the cardiovascular and nervous systems. YC-1 and
BAY 58-2667 represent new classes of activators of catalytic domain, a central part, and an N-terminal region.
NO-sensitive GC. The catalytic C-terminal domains of each subunit of NO-
sensitive GC display the highest degree of homology.32 These
versus membrane-bound) but also in their structure and domains are also very similar to the respective regions in the
regulation (see review15). The membrane-bound, peptide-ac- peptide-activated, membrane-bound guanylyl cyclases and in
tivated GCs are structurally related to the cytosolic enzymes the adenylyl cyclases (AC). The catalytic domains of AC and
but are not stimulated by NO. These GCs belong to the group NO-sensitive GC are structurally closely related as shown by
of receptor-linked enzymes containing one membrane- the functional expression of AC/GC heterodimers33 and the
spanning region. For detailed review of this GC family, see conversion of nucleotide specificity by the mutation of only
the article “Guanylyl Cyclase Receptors” in this series. three amino acids.34 In the crystal structure obtained for these
AC domains,35,36 the binding site for the activator forskolin
Isoforms and Structure of NO-Sensitive GC has been identified. The analogous region in NO-sensitive
Purification of NO-sensitive GC from tissues of various GC may be of special interest with regard to the possible
organisms such as rat and bovine lung and brain was achieved binding site of the activator YC-1 (see later section).
by several groups. Subsequently, the ␣1 and ␤1 subunits with The central regions preceding the catalytic domains show
molecular masses of 73 and 70 kDa, respectively, were considerable homology to the membrane-bound enzymes.37
cloned and sequenced.16 –19 The so-called ␣3 and ␤3 subunits In analogy, these regions are probably involved in the
of NO-sensitive GC20 represent human variants of the ␣1 and dimerization of the subunits, although detailed investigation
␤1 subunits rather than different isoforms, and changes in the is still missing.
reading frame account for the differences in amino acid The N-terminal regions of the subunits of NO-sensitive GC
sequences.21 comprise the heme-binding domain. The prosthetic heme
Homology screening yielded another subunit of NO- group mediates the NO-induced stimulation subsequent to
sensitive GC, ␣2.22 The occurrence of the ␣2 subunit was binding NO. A histidine residue (His-105) within this region
demonstrated on the protein level in human placenta, and the of the ␤1 subunit was shown to bind to the heme iron, thereby
␤1 subunit was identified as the dimerizing partner.23 Despite acting as the proximal ligand. Deletion of His-105 has been
little sequence homology between the two ␣ subunits in the shown to abrogate NO activation of the enzyme.38
N-terminal domains, no differences in catalytic activity, NO
stimulation, or substrate affinity could be detected between Prosthetic Heme Group
the ␣1␤1 and the ␣2␤1 heterodimers. Recent data suggest that NO-sensitive GC is a hemoprotein and presence of the
the C-terminal peptide of the ␣2 subunit defines the particular prosthetic heme group is mandatory for the activation of the
property of this subunit: the ability to interact with PDZ enzyme by NO.39,40 Removal of the heme group abolishes
domains. Through the interaction with the PDZ domain– NO-induced activation, which can be restored after reconsti-
containing protein PSD-95, the ␣2␤1 heterodimer was shown tution of NO-sensitive GC with heme.41,42 The heme stoichi-
to be localized to synaptic membranes.24 In accordance with ometry has been agreed to be one mole per mole GC
a putative role in synaptic transmission, the highest expres- heterodimer. The absorbance maximum at 430 nm is indica-
sion of the ␣2 subunit was found in brain. Furthermore, the ␤1 tive for a five-coordinated ferrous heme with a histidine as the
subunit was detected at the presynaptic membrane of CA1 axial ligand.43 In contrast to other hemoproteins such as

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98 Circulation Research July 25, 2003

hemoglobin or myoglobin, the heme of NO-sensitive GC vivo studies, differ in parameters like NO release or thiol
does not bind oxygen.1,44 requirement. For this reason, EC50 values reported for these
Both, the ␣ and ␤ subunits are required for proper binding substances vary considerably depending on the donor and the
and orientation of the heme group. Sequential truncation assay system used. Whereas NO donors such as GSNO or
showed that the subunits contribute unequally to heme SNP have to be used at micromolar concentrations for
binding with the ␤1 subunit playing a more important role45; half-maximal activation, the frequently used compound
in addition, homodimers of the N-terminal part of the ␤1 DEA-NO, which releases NO spontaneously, shows an EC50
subunit (the first 385 amino acid) expressed in bacteria were value of approximately 300 nmol/L.24,57
shown to bind heme in a manner similar to the wild-type
enzyme.46 Mechanism of Activation
The His-105 of the ␤1 subunit was identified as the heme Among the three redox forms of NO (NO⫺, NO•, NO⫹), only
coordinating residue.38,47 Mutation of this histidine led to the the uncharged NO radical (NO · ) has been shown to signifi-
generation of an NO-insensitive, heme-depleted enzyme with cantly activate NO-sensitive GC.58 Activation of the enzyme
intact basal activity. In addition, two conserved cysteines by NO involves the binding to the enzyme’s prosthetic heme
adjacent to the His-105 on the ␤1 subunit appear to play a role group, which leads to the up to 200-fold activation of the
in the formation of the proper heme pocket. Mutation of these enzyme.43,59
residues led to loss of enzyme-bound heme and NO respon- The heme group of NO-sensitive GC exhibits an absor-
siveness, which could be regained after heme reconstitution.48 bance maximum at 431 nm, which is indicative of a five-
coordinated ferrous heme with a histidine as the axial ligand
Activation of NO-Sensitive GC by NO at the fifth coordinating position.43 Several models exist to
Already in the 1970s, NO-releasing compounds were found explain the activation of NO-sensitive GC, the simplest is
to be potent activators of NO-sensitive GC.49,50 Similarly, thought to occur in two steps: binding of NO to the sixth
nitrovasodilators like glyceroltrinitrate or isosorbid dinitrate coordination position of the heme results in a six-coordinated
therapeutically used for the treatment of coronary heart NO-Fe2⫹-His-complex. The subsequent breakage of the
disease act by stimulation of the enzyme. These nitrates do histidine-to-iron bond leads to the formation of a five-
not release NO spontaneously; instead, they have to undergo coordinated nitrosyl-heme complex with an absorbance max-
bioactivation that either yields NO or nitrosothiols. imum at 398 nm. The opening of the histidine-to-iron bond is
Despite the stimulatory effect of these NO-containing considered to initiate a conformational change resulting in the
compounds, the physiological significance of NO-induced activation of the enzyme. In support of this simple model,
activation of the enzyme did not become apparent until the protoporphyrin IX activates NO-sensitive GC independently
identification of endothelium-derived relaxing factor (EDRF) of NO.60 Protoporphyrin IX, the iron-free precursor of heme,
as NO.51,52 Formation of EDRF had been shown to occur in structurally resembles the NO-heme complex in which the
endothelial cells in response to vasodilatory agonists such as iron is moved out of the plane of the porphyrin ring. Thus, in
acetylcholine, histamine, or bradykinin, leading to vasodila- both structures, the axial histidine is unbound. However, the
tion via the activation of NO-sensitive GC in smooth muscle release of the histidine-iron bond appears not to be sufficient
cells.53 After the discovery of NO in the vascular system, NO for the activation of NO-sensitive GC as a mutant lacking the
formation was reported to occur throughout the body.54 The proximal histidine did not show an increased catalytic rate.
enzymes responsible for the synthesis of NO were identified Therefore, the proximal histidine not only coordinates the
and termed NO synthases of which three isoforms are known heme group but also mediates the stimulatory action.
to date. The inducible isoform produces relatively high NO Recently, a more complex model of NO activation has
concentrations and thereby exhibits direct toxic effects. The been introduced. By using stopped-flow spectroscopy, Zhao
enzyme is mainly expressed in macrophages and plays a role et al61 investigated the subsecond kinetics of NO activation of
in the nonspecific immune response. The neuronal and NO-sensitive GC and provided data that the transition from
endothelial NO synthases are constitutively expressed en- the six-coordinate to the five-coordinate complex depended
zymes that are regulated by the intracellular calcium concen- on the NO concentration present. This results in a model
tration. The endothelial NO synthase is also activated by where NO not only activates the enzyme by binding to the
Akt/PKB-dependent phosphorylation triggered by shear heme but also regulates the velocity of activation via binding
forces exerted on the endothelial cells.55 Both isoforms to a second non-heme binding site. However, Bellamy et al,62
produce relatively low NO concentrations (1 to 100 nmol/L). reinterpreted the data by Zhao et al in favor of the simple
At these low concentrations, NO functions as a signaling two-state model and claimed the postulated additional NO
molecule, and most of its effects are mediated via the site as unnecessary. These two models are currently disputed
activation of NO-sensitive GC. between the two groups.62,63
In fact, an EC50 value for NO-sensitive GC as low as 2
nmol/L NO has been determined in cerebellar cell suspen- Deactivation of NO-Sensitive GC
sions using a technique to deliver constant concentrations.56 In an early study, Palmer et al51 showed that NO/EDRF-
Thus, activation of NO-sensitive GC in intact cells can be induced relaxation of aortic rings could be reinduced already
achieved at concentrations that are below those potentially 1 to 2 minutes after the first stimulus, indicating fast deacti-
inhibitory for other cellular targets such as mitochondrial vation of NO-sensitive GC in the biological system. Further
cytochromes.56 NO donors, commonly used in in vitro and in studies on NO dissociation and deactivation have been

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Friebe and Koesling Regulation of NO-Sensitive Guanylyl Cyclase 99

Maximal Stimulation of NO-Sensitive GC by Different Classes


of Activators
GC Activator Maximal Stimulation
NO ⬇200-fold
YC-1 ⬇10-fold
YC-1⫹submaximal NO ⬇200-fold
BAY 58–2667 ⬇40-fold
BAY 58–2667 ⬇200-fold (heme-free enzyme)

concentrations.72 Therefore, ODQ represents an important


tool to discriminate cGMP-dependent and cGMP-
independent effects of NO. Inhibition of NO-sensitive GC by
ODQ has been demonstrated in a variety of other cells and
tissues.71,73–75 Studies with purified NO-sensitive GC re-
vealed that ODQ binds in an NO-competitive manner and
leads to an apparently irreversible inhibition of the stimulated
enzyme leaving basal activity unchanged. Spectral analysis
suggests oxidation of the heme iron as underlying mechanism
of the inhibitory effect.76,77 The notion that the heme iron is
Figure 2. Pharmacological compounds acting on NO-sensitive the target of ODQ is confirmed by the finding that ODQ is not
GC. YC-1 and BAY 58-2667 are novel activators of NO-sensitive
GC that increase cGMP production independent of NO. YC-1
able to inhibit the stimulation of NO-sensitive GC induced by
potentiates the stimulatory action of NO and thus serves as “NO the iron-free protoporphyrin IX.78 A derivative of ODQ, NS
sensitizer”; BAY 58-2667 preferentially activates the heme- 2028 (oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one), has been
depleted form of the enzyme. ODQ, in contrast, inhibits published to have properties similar to those of ODQ.79
NO-sensitive GC by oxidizing the heme iron leaving basal activ-
ity intact. Other inhibitors such as methylene blue and LY-83583
show less specificity than ODQ and, eg, inhibit the olfactory
performed with purified enzyme, cytosolic fractions, and cyclic nucleotide-gated ion channel80 or interfere with NO
intact cells. formation and NO release from NO synthases81; therefore,
NO dissociation measured spectrophotometrically resulted ODQ should be preferred as specific GC inhibitor.
in a half-life of the NO-GC complex of approximately 2
minutes, which was accelerated to a half-life of ⬇5 seconds Novel Activation Mechanisms of
by the addition of the substrate MgGTP.64,65 This accelerating NO-Sensitive GC
effect of GTP is corroborated by a study using resonance Besides the well-established NO/heme-mediated stimulation,
Raman spectroscopy, which suggested binding of GTP in the two novel mechanisms of activation have been identified for
proximity of bound NO, thereby possibly regulating NO NO-sensitive GC, indicating considerable therapeutic poten-
binding.66 A half-life of approximately 3 minutes but no tial (Table). First, the activator YC-1 (see Figure 2) was
increase in the NO dissociation rate on the addition of shown to act as an “NO sensitizer,” greatly enhancing the
MgGTP was shown in a different spectrophotometric study.67 sensitivity of GC toward NO.82,83 Derivatives of YC-1 and
The half-life of approximately 5 seconds for the NO-GC other novel substances have been identified84 – 87 (for
complex was confirmed monitoring deactivation, ie, the structure-function relationship, see Selwood et al88). By
decline in cGMP forming activity of the purified enzyme68 or increasing the NO responsiveness under pathological condi-
in cytosolic preparations of retina.69 Measurements in intact tions characterized by reduced NO release, these NO sensi-
cerebellar cells showed an even 10-fold faster deactivation tizers may become important drugs in the treatment of
with a half-life of 0.2 seconds.70 In conclusion, deactivation various cardiovascular diseases. Moreover, with the sub-
of NO-sensitive GC was shown to be in the range required for stance BAY 58-2667 (see Figure 2), another mode of GC
rapid signaling in various systems. Additional research is activation has been described recently. This substance was
necessary to unravel the special feature of the heme group of reported as an NO-independent activator whose effect was
NO-sensitive GC permitting such fast NO dissociation. even more pronounced on the heme-free enzyme.89 Both
YC-1 and BAY 58-2667 have been shown to exert beneficial
Inhibitors effects in the cardiovascular system based on their antiplatelet
Several substances have been found to inhibit NO-sensitive and vasodilatory activity.89 The effects of both classes of
GC. The quinoxalin derivative 1H-[1,2,4]oxadiazolo[4,3-a]- activators on NO-sensitive GC will be discussed in the
quinoxalin-1-one (ODQ; Figure 2) was shown to be a potent following section.
and selective inhibitor of NO-sensitive GC in brain slices.71 YC-1 (3-(5⬘-hydroxymethyl-2⬘-furyl)-1-benzylindazole)90
ODQ does not lead to the inhibition of the membrane-bound was the first compound of this series to be identified.
guanylyl or adenylyl cyclases and interferes with the stimu- Originally, YC-1 had been published as an activator of
lation of NO synthase and other heme proteins only at high NO-sensitive GC in rabbit and human platelets leading to an

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100 Circulation Research July 25, 2003

increase in the intracellular cGMP concentration.91,92 YC-1 occurrence of the heme-free form of NO sensitive GC under
causes a 10-fold activation of purified NO-sensitive GC physiological conditions remains to be elucidated. Photoaf-
independent of NO.82,93 Despite this NO independence, YC-1 finity labeling and binding studies suggest binding of the
failed to stimulate the heme-free enzyme, showing that the compound to both subunits of NO-sensitive GC.
effect of YC-1 required the presence of the prosthetic heme
group. Most interestingly, YC-1 sensitized NO-sensitive GC Sensitization/Desensitization Within
toward NO as YC-1 shifted the EC50 for NO to the left by one NO/cGMP Signaling
order of magnitude.82,93 Mechanistically, YC-1 was shown to In the cardiovascular system, NO/cGMP signaling is important
slow down deactivation of NO-stimulated GC, which is in the regulation of vascular tone and primary hemostasis. As
sufficient to explain the sensitizing effect of YC-1.68,83 Rather NO-sensitive GC occurs mainly in smooth muscle cells and
unexpectedly, YC-1 also turned CO into an effective activa- platelets, many studies have been performed in either cell type,
tor, despite the fact that by itself, CO is hardly able to activate and NO sensitivity has been shown to vary. This section
the purified enzyme.82,94 concentrates on the modulation of NO-sensitive GC and beyond
By spectral analysis and enzyme studies, YC-1 was shown in the cGMP signaling cascade; the broader phenomenon of NO
to bind to a site different from the heme group, suggesting a tolerance, which also includes NO bioavailability, is being
so far unknown allosteric site on the enzyme.83 Controversial covered in another article of this series.
reports exist on the localization of this allosteric site. In order Sensitization/desensitization describe an alteration of the
to identify this site, Stasch et al95 used a novel high-affinity NO/cGMP response toward a given NO challenge. This
YC-1 analogue, BAY 41-2272, as photoaffinity label. An process may be caused by a change in enzyme reactivity, eg,
N-terminal region of the ␣1 subunit (amino acids 236 to 290) by posttranslational modification, or an alteration of the
was identified to comprise the YC-1 binding site. However, amount of the enzyme present, the latter being discussed in
this region is not conserved in the ␣2 subunit albeit the the next section. Sensitization/desensitization of the cGMP
␣2-containing heterodimer exhibits virtually identical sensi- response has been mostly attributed to a changed responsive-
tivity toward YC-1.23 In theory, the structural similarity ness of NO-sensitive GC. However, the impact of cGMP
between AC and GC in combination with the sensitizing degradation has not been examined in most cases.
effect of the respective activators forskolin and YC-1 suggest First reports on an increased sensitivity of the NO-induced
the C-terminal catalytic region as the YC-1 binding site (see cGMP response have been performed on isolated blood
previous section); this notion is supported by different muta- vessels that were either endothelium-denuded or in which
tions within this region of GC either mimicking or abolishing endothelial NO production was impaired by inhibitor treat-
the effects of YC-1.68,96 Nevertheless, definitive conclusions ment.106 –108 In all these studies, removal of NO led to an
about the allosteric site involved in NO sensitization of the increase in the potency of vasodilators used for relaxation.
enzyme await protein crystallography. These in vitro data were corroborated in in vivo experiments
The effects of YC-1 on NO/cGMP signaling have been by Moncada et al109 who found an enhanced antihypertensive
investigated in a variety of cells and tissues. In vascular smooth response to glyceryltrinitrate in rats treated with inhibitors of
muscle cells, YC-1 was reported to increase cGMP levels and to eNOS. This “supersensitive” state did not require long-term
induce a concentration-dependent relaxation of endothelium- treatment but was also detected in vessels treated with
denuded rat aortic rings.93,97,98 Other tissues in which YC-1 L-NAME for only 15 minutes. More recent reports show
induced cGMP elevations include guinea pig trachea,99 guinea similar results in mice lacking the endothelial NO synthase.110
pig colon,100 corpus cavernosum,101 and pig urethra.102 In endo- Thus, reduction of the NO concentration in vitro or in vivo leads
thelial cells, the YC-1 induced cGMP increases were most to an increase in sensitivity of the signaling pathway.
pronounced as in these cells YC-1 did not only stimulate Vice versa, decreased sensitivity of the NO/cGMP system
NO-sensitive GC directly but also potentiated the stimulatory has been demonstrated under various conditions after treat-
effect of the endogenously synthesized NO.103 ment with NO-releasing agents.111–113 Transgenic mice over-
In platelets, YC-1 was shown to inhibit adhesion and aggre- expressing eNOS showed a decreased response toward endo-
gation.92,104,105 In these cells, YC-1 led to a drastic, over thelium-dependent and -independent vasorelaxation, which
1000-fold increase in cGMP in the presence of NO. Closer was attributed to a decreased activity of NO-sensitive GC.114
analysis revealed that YC-1 inhibited various PDEs.98,105 Due to In a recent report, Bellamy et al115 showed a rapid NO-
the high catalytic rate of PDEs, some of the YC-1 effects induced desensitization of NO-sensitive GC within seconds.
observed in intact cells may be caused by the inhibition of PDEs Despite these reports, factors responsible for sensitization/
rather than solely by the stimulation of NO-sensitive GC. desensitization of NO-sensitive GC have not been identified
A second novel mechanism of activation has been shown so far (for phosphorylation, see next section). Although
to occur using the substance BAY 58-2667.89 This com- regulation of NO-sensitive GC by thiol-modifying or redox-
pound, an amino dicarboxylic acid, alone led to a moderate active substances like superoxide has been postulated for
activation of NO-sensitive GC; this stimulation was additive more than 30 years, the functional consequences have not
to that of NO, which is in contrast to the potentiation seen been unequivocally demonstrated on the purified enzyme,
with YC-1. Surprisingly, BAY 58-2667 stimulated the heme- and the underlying molecular modifications are unknown.
oxidized or heme-depleted purified enzyme almost 200-fold. Theoretically, NO sensitivity of GC could be controlled by
This indicates that BAY 58-2667 may be able to substitute for the heme content of the enzyme. However, the heme/protein
the heme group or stabilize the heme-free conformation; the ratio of 1 mole per mole obtained in several purification

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Friebe and Koesling Regulation of NO-Sensitive Guanylyl Cyclase 101

demonstrated by many groups. Variation of GC expression is


conceivable in order to adapt to the amount of NO available
under certain physiological or pathophysiological conditions.
As a matter of fact, the development of nitrate tolerance may
be based in part on downregulation of NO-sensitive GC. In
line with this reasoning, various NO donors were reported to
decrease the mRNA and protein levels of NO-sensitive GC in
pulmonary artery cells by a cGMP-dependent mechanism.121
However, NO tolerance in humans was not paralleled by a
changed GC content122 and, paradoxically, increased expres-
sion of both GC subunits was detected in blood vessels from
NO-tolerant rats and rabbits.123 It should be noted that both
studies reported on a reduced phosphorylation of the down-
stream target of cGMP-dependent kinase, VASP. The re-
Figure 3. Feedback inhibition within the NO/cGMP signaling duced VASP phosphorylation under the condition of NO
cascade in platelets. Increased cGMP levels lead to the direct tolerance can be interpreted as switched-off NO/cGMP sig-
activation of phosphodiesterase 5, which in turn degrades naling caused by PDE activation (see earlier sections). There
cGMP more rapidly. Phosphorylation of phosphodiesterase 5 by
cGMP-dependent protein kinase increases its cGMP affinity.
is a recent article on post-transcriptional regulation of the ␣1
This feedback mechanism regulates the sensitivity of the subunit of NO-sensitive GC in rat aorta.124 The authors
NO/cGMP signaling cascade in platelets. identified HuR (Human R, embryonic lethal abnormal visual
[ELAV]–like RNA-binding protein) as a factor stabilizing
procedures from different tissues argues against a varying GC ␣1 mRNA. YC-1–induced activation of GC decreased
heme content. Besides, mice with limited heme synthesis HuR expression, thereby inducing rapid degradation of GC ␣1
were shown to contain a normally functioning NO-sensitive mRNA and lowering ␣1 subunit expression as a negative
GC.116 Thus, a molecular mechanism for sensitization/desen- feedback response.
sitization of NO-sensitive GC is still lacking. Expression of GC subunits was also studied under condi-
In most of these cases, sensitization/desensitization of the tions of low NO availability. Although animals in which NO
cGMP response was assessed by measuring intracellular production was impaired either by gene knockout110 or NO
cGMP levels not taking into account the downstream cGMP- synthase inhibitor treatment125 showed an enhanced sensitiv-
degrading activity of phosphodiesterases (PDE). Recent re- ity toward additional NO, aortic GC content was not found to
sults on cGMP-induced stimulation of PDE5 provide another be altered.110
possibility to explain the sensitization/desensitization within Besides the availability of NO, the effects of various other
NO/cGMP signaling. In platelets, the NO-induced cGMP conditions on GC expression have been studied. Hyperten-
response was fast (less than a minute) and controlled by sion and ageing appeared to be factors negatively influencing
PDE5 activity.117 Desensitization did not occur on the level of the expression of GC.126 –129 High salt intake by spontane-
NO-sensitive GC, which remained fully active but was ously hypertensive rats led to a reduced vascular GC content
attributed to an increased activity of PDE5. PDE5 is directly in the aorta, which was paralleled by impairment of the
activated by cGMP binding to one of the enzyme’s regulatory vascular relaxation response to NO donors.130 Various addi-
GAF domains (GAF-A).118 Surprisingly, PDE5 activation tional factors like cytokines, estradiol, and ␤-amyloid pep-
was very sustained and still observed 1 hour after the removal tides appear to reduce GC protein.131–134 Yet, also upregula-
of NO.119 PDE5 activation was paralleled by phosphorylation tion of aortic NO-sensitive GC in Wistar rats after myocardial
by the cGMP-dependent protein kinase I, which increases the infarction was reported.135
cGMP affinity of the GAF-A domain.120 The exact mecha-
nism of cGMP-induced activation of PDE5 is outlined in Putative Activators or Inhibitors
more detail in another article of this series. In sum, there is The issue of CO being a physiologically relevant activator of
evidence for a new, so far unknown, feedback inhibition NO-sensitive GC has not convincingly been solved yet. CO
within NO/cGMP signaling (Figure 3). Within this feedback was first shown to activate NO-sensitive GC approximately
loop, cGMP bound to the regulatory GAF domain of PDE5 4-fold in platelet supernatant.136 Functionally, CO has been
serves as a memory for the amount of NO the cell has been reported to influence smooth muscle tone, platelet aggrega-
exposed to adjusting the subsequent cGMP response toward tion, and neuronal signaling. CO is endogenously produced
further incoming NO signals. Further experimental evidence by the enzyme family of heme oxygenases in the process of
is required to find out whether PDE5 activation is a general heme degradation. The mechanism by which CO acts on
mechanism explaining the above described changes in the NO-sensitive GC appears similar to that of NO: due to the
sensitivity of NO/cGMP signaling. high affinity for heme, CO binds to NO-sensitive GC under
the formation of a hexa-coordinated carbonyl-heme com-
Regulation of the Expression of plex.43,137 However, in contrast to NO, CO does not induce
NO-Sensitive GC the release of the histidine-to-iron bond. As breakage of this
Parallel to the changed responsiveness of the cGMP signaling bond is prerequisite for GC activation, the lack of the
system, expressional regulation of NO-sensitive GC has been transformation of the six- to the five-coordinated complex

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102 Circulation Research July 25, 2003

explains the rather poor GC-activating property of CO. which may reflect the association to the neuronal and endo-
Results with the novel GC activator YC-1 revitalized the idea thelial NO synthases, respectively.
of CO as an endogenous activator of NO-sensitive GC. In the Furthermore, besides the physiological and pharmacologi-
presence of YC-1, CO stimulates NO-sensitive GC to the cal NO-based activation, two so far unknown routes to
same extent as NO.82,94 Yet, a possible physiological role of sensitize and stimulate the enzyme have been presented. The
CO would require not only rather high CO concentrations but modulation of the NO responsiveness of NO-sensitive GC
also an endogenously occurring YC-1–like substance. So far, has important pharmacological implications for the therapy of
evidence for such an endogenously occurring YC-1–like cardiovascular diseases. The relevance of the respective
substance is missing. Furthermore, CO was suggested to substances has been already been substantiated in various
negatively regulate NO stimulation of GC by competing for animal models. A better understanding of the underlying
the heme group; however, this suggested inhibitory role molecular mechanisms will greatly improve our current
appears unlikely because CO would have to be available at knowledge of the enzyme, which in turn may also help to
extremely high intracellular concentrations based on the over elucidate the physiology and pathophysiology of NO/cGMP
1500-fold lower heme affinity compared with NO.138 signaling.
Before the identification NO as of the physiological stim-
ulator of NO-sensitive GC, a multitude of mostly redox- Acknowledgments
active substances have been proposed as activators (see This work was supported by the Deutsche Forschungsgemeinschaft
review15). Several mechanisms of activation have been pos- DFG 1157/3-2.
tulated and subsumed as “redox regulation.”139 With respect
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Regulation of Nitric Oxide-Sensitive Guanylyl Cyclase
Andreas Friebe and Doris Koesling

Circ Res. 2003;93:96-105


doi: 10.1161/01.RES.0000082524.34487.31
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