Nitric oxide-sensitive guanylyl cyclase: structure and regulation

By the formation of the second messenger cGMP, NO-sensitive guanylyl cyclase (GC) plays a key role within the NO/cGMP signaling cascade which participates in vascular regulation and neurotransmission. The enzyme contains a prosthetic heme group that acts as the acceptor site for NO. High affinity binding of NO to the heme moiety leads to an up to 200-fold activation of the enzyme. Unexpectedly, NO dissociates with a half-life of a few seconds which appears fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogs act as NO sensitizers and led to the discovery of a novel pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme. The two isoforms of the heterodimeric enzyme (alpha1beta1, alpha2beta1) are known that are functionally indistinguishable. The alpha2beta1-isoform mainly occurs in brain whereas the alpha1beta1-enzyme shows a broader distribution and represents the predominantly expressed form of NO-sensitive GC. Until recently, the enzyme has been thought to occur in the cytosol. However, latest evidence suggests that the alpha2-subunit mediates the membrane association of the alpha2beta1-isoform via interaction with a PDZ domain of the post-synaptic scaffold protein PSD-95. Binding to PSD-95 locates this isoform in close proximity to the NO-generating synthases thereby enabling the NO sensor to respond to locally elevated NO concentrations. In sum, the two known isoforms may stand for the neuronal and vascular form of NO-sensitive GC reflecting a possible association to the neuronal and endothelial NO-synthase, respectively.     

A functional domain of the alpha1 subunit of soluble guanylyl cyclase is necessary for activation of the enzyme by nitric oxide and YC-1 but is not involved in heme binding

Soluble guanylyl cyclase is a heterodimeric enzyme consisting of an alpha(1) and a beta(1) subunit and is an important target for endogenous nitric oxide and the guanylyl cyclase modulator YC-1. The activation of the enzyme by both substances is dependent on the presence of a prosthetic heme group. It has been unclear whether this prosthetic heme group is sandwiched between the alpha(1) and beta(1) subunits or whether it exclusively binds to the beta(1) subunit. Here we analyze progressive amino-terminal deletion mutants of the human alpha(1) subunit after co-expression with the human beta(1) subunit in the baculovirus/Sf9 system. Spectral, biochemical, and pharmacological analysis shows that the first 259 amino acids of the alpha(1) subunit can be deleted without loss of sensitivity to nitric oxide (NO) or YC-1 or loss of heme binding of the respective enzyme complex with the beta(1) subunit. This is in contrast to previous data indicating that NO sensitivity and a functional heme binding site requires full-length amino termini of bovine alpha(1) and beta(1) subunits. Further deletion of the first 364 amino acids of the alpha(1) subunit leads to an enzyme complex with preserved heme binding but loss of sensitivity to NO or YC-1 despite induction of the typical spectral shift by NO binding to the prosthetic heme group. We conclude that 1) the amino-terminal part of the alpha(1) subunit is not involved in heme binding and 2) amino acids 259-364 of the alpha(1) subunit represent an important functional domain for the transduction of the NO activation signal and likely represent the target for NO-sensitizing substances like YC-1.     

Guanylyl cyclase: NO hits its target

The NO receptor, NO-sensitive guanylyl cyclase, plays a key role in the NO/cGMP signal-transduction cascade. Two isoforms of the enzyme are currently known, the widely distributed vascular alpha1beta1 isoform and the neuronal alpha2beta1 isoform predominantly expressed in brain. Interaction with the PSD-95 (postsynaptic density protein-95) family of scaffolding proteins targets the neuronal alpha2beta1 isoform to synaptic membranes. The NO sensor of the guanylyl cyclase is formed by the prosthetic haem group, where NO binding takes place and induces the up to 200-fold activation of the enzyme. The haem group allows tight regulation of enzymic activity by NO and represents the most striking feature of the enzyme, as it differs in many aspects from the well-characterized haem groups of other haemoproteins. The new NO sensitizers such as YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] affect activation by NO and CO by mechanisms that are currently subject to intense research.     

Elongation factor 2 as a target for selective inhibition of protein synthesis in vitro by the novel aromatic biasamidine

By the formation of cGMP the NO-sensitive guanylyl cyclase plays a key role within the NO/cGMP signaling cascade involved in vascular regulation and neurotransmission. The prosthetic heme group of the enzyme acts as the NO sensor, and binding of NO induces conformational changes leading to an up to 200-fold activation of the enzyme. The unexpected fast dissociation half-life of NO of a few seconds is fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogues acting as NO sensitizers uncovered a new pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme.Two existing isoforms of the heterodimeric guanylyl cyclase (₁₁, ₂₁) are known that are functionally indistinguishable. Up to now, the NO-sensitive guanylyl cyclase has been considered as a soluble enzyme. However, recent evidence about the ₂₁ isoform interacting with a PDZ domain of the postsynaptic scaffold protein PSD-95 suggests that the ₂ subunit directs a membrane association of this isoform. The interaction with PSD-95 locates the ₂₁ isoform in close proximity to the NO-generating NO synthase thereby enabling the NO sensor to respond to locally raised NO concentrations.     

Functional characterization of nitric oxide and YC-1 activation of soluble guanylyl cyclase: structural implication for the YC-1 binding site?

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme formed by an alpha subunit and a beta subunit, the latter containing the heme where nitric oxide (NO) binds. When NO binds, the basal activity of sGC is increased several hundred fold. sGC activity is also increased by YC-1, a benzylindazole allosteric activator. In the presence of NO, YC-1 synergistically increases the catalytic activity of sGC by enhancing the affinity of NO for the heme. The site of interaction of YC-1 with sGC is unknown. We conducted a mutational analysis to identify the binding site and to determine what residues were involved in the propagation of NO and/or YC-1 activation. Because guanylyl cyclases (GCs) and adenylyl cyclases (ACs) are homologous, we used the three-dimensional structure of AC to guide the mutagenesis. Biochemical analysis of purified mutants revealed that YC-1 increases the catalytic activity not only by increasing the NO affinity but also by increasing the efficacy of NO. Effects of YC-1 on NO affinity and efficacy were dissociated by single-point mutations implying that YC-1 has, at least, two types of interaction with sGC. A structural model predicts that YC-1 may adopt two configurations in one site that is pseudosymmetric with the GTP binding site and equivalent to the forskolin site in AC.     

A-350619: a novel activator of soluble guanylyl cyclase

Nitric oxide (NO) is a key mediator in many physiological processes and one of the major receptors through which NO exerts its effects is soluble guanylyl cyclase. Guanylyl cyclase converts GTP to cyclic GMP as part of the cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, inhibition of platelet aggregation and immune response. The properties of A-350619, a novel soluble guanylyl cyclase activator, were examined to determine the modulatory effect on the catalytic properties of soluble guanylyl cyclase. A-350619 increased V(max) from 0.1 to 14.5 micromol/min/mg (145 fold increase), and lowered K(m) from 300 to 50 microM (6 fold decrease). When YC-1 (another sGC activator) and A-350619 were combined, a 156 fold increase in V(max) and a 5 fold decrease in Km were observed, indicating that the modulation of the enzyme brought about by YC-1 and A-350619 are not additive, suggesting a common binding site. Activation of soluble guanylyl cyclase by A-350619 was partially inhibited by ODQ, a specific inhibitor of soluble guanylyl cyclase by oxidation of the enzyme heme. YC-1 and A-350619 after pre-treatment with N-omega-nitro-L-arginine, an NO-synthase inhibitor, relaxed cavernosum tissue strips in a dose-dependent manner with EC(50) of 50 microM and 80 microM, respectively. Addition of SNP potentiated the relaxation effect of YC-1 and A-350619, shifting the dose-response curve to the left to 3 microM and 10 microM, respectively. Consistent with its biochemical activity, A-350619 (1 micromol/kg) alone induced penile erection in a conscious rat model. Activation of soluble guanylyl cyclase in cavernosum tissue as an alternate method of enhancing the effect of NO may provide a novel treatment of sexual dysfunction.     

Studying the structure and regulation of soluble guanylyl cyclase

Soluble guanylyl cyclase acts as the receptor for the signaling molecule nitric oxide. The enzyme consists of two different subunits. Each subunit shows the cyclase catalytic domain, which is also conserved in the membrane-bound guanylyl cyclases and the adenylyl cyclases. The N-terminal regions of the subunits are responsible for binding of the prosthetic heme group of the enzyme, which is required for the stimulatory effect of nitric oxide (NO). The five-coordinated ferrous heme displays a histidine as the axial ligand; activation of soluble guanylyl cyclase by NO is initiated by binding of NO to the heme iron and proceeds via breaking of the histidine-to-iron bond. Recently, a novel pharmacological and possibly physiological principle of guanylyl cyclase sensitization was demonstrated. The substance YC-1 has been shown to activate the enzyme independent of NO, to potentiate the effect of submaximally effective NO concentrations, and to turn carbon monoxide into an effective activator of soluble guanylyl cyclase.     

Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin.

L-Arginine-derived nitric oxide acts as an inter- and intracellular signal molecule with cytosolic guanylyl cyclase as the effector system. Two NO synthase isoenzymes are postulated: a cytokine-inducible enzyme in macrophages and a constitutive, Ca2(+)-regulated enzyme in various other cells. An NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2',5'-ADP-Sepharose. The enzyme was identified as an NO synthase with a specific NO-chemiluminescence method and with purified cytosolic guanylyl cyclase as an NO-sensitive detection system. The purified NO synthase was, besides Ca2+/calmodulin and NADPH, largely dependent on tetrahydrobiopterin as a cofactor.     

Rate of deactivation of nitric oxide-stimulated soluble guanylate cyclase: influence of nitric oxide scavengers and calcium

Soluble guanylate cyclase (sGC) is highly activated by nitric oxide (NO) and is the known mediator of the effects of NO on a variety of physiological processes. The rates at which sGC is activated and deactivated are therefore of wide interest since they determine the duration of a tissue's response to NO. The effect of NO on smooth muscle dissipates in 1-2 min, suggesting that both activation and deactivation are fast. In vitro measurements show that the activation of sGC occurs in less than a second, while the deactivation takes several hours at 20 degrees C. However, recent reports indicate that Mg-GTP, oxyhemoglobin, and reducing and oxidizing agents could deactivate the cyclase in several seconds to minutes, though the effectiveness of each of these agents is in dispute. We investigated the lifetime of NO-sGC in the cytosol of retina by monitoring its enzymatic activity at 20 degrees C. Our results show that Mg-GTP, the substrate of NO-sGC, has no influence on the deactivation. Similarly, reducing agents glutathione and dithiothreitol shortened the half-life of NO-sGC only by about 30%. The greatest effect on the deactivation was caused by scavengers of NO: oxyhemoglobin reduced the half-life of NO-sGC from 106 min to 18 s; another NO scavenger, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), reduced it to 42 s (20 degrees C). Similarly rapid deactivation was observed with the enzyme from bovine lung, immunoprecipitated enzyme from bovine retina, and heme-deficient enzyme from bovine retina reconstituted with heme. On the other hand, YC-1, an activator of sGC, stabilized the activated enzyme, preventing NO dissociation, as was evident from the inability of oxyhemoglobin or CPTIO to deactivate NO-sGC. Calcium, which is known to inhibit NO-sGC, also inhibited the effects of oxyhemoglobin and CPTIO, slowing down the deactivation of the enzyme. Lithium, which is also known to inhibit NO-sGC, had no effect on the deactivation rate of the enzyme. These results, taken together, suggest that two factors with major impact on the lifetime of NO-sGC are the proximity to NO scavengers and the calcium concentration in the cell.     

Nitric oxide activates the beta 2 subunit of soluble guanylyl cyclase in the absence of a second subunit.

Previously characterized mammalian soluble guanylyl cyclases form alpha/beta heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named alpha(1), beta(1), alpha(2), and beta(2). The alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzyme isoforms have been rigorously characterized. The role of the beta(2) subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the beta(2) subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the beta(3) subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mm free Mn(2+). The EC(50) values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the alpha(1)/beta(1) heterodimeric form. In the presence of the detergent Tween, NO sensitivity of beta(2) was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the beta(2) subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.     

Post-transcriptional regulation of soluble guanylyl cyclase expression in rat aorta

We investigated the molecular mechanism of cyclic GMP-induced down-regulation of soluble guanylyl cyclase expression in rat aorta. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), an allosteric activator of this enzyme, decreased the expression of soluble guanylyl cyclase alpha(1) subunit mRNA and protein. This effect was blocked by the enzyme inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) and by actinomycin D. Guanylyl cyclase alpha(1) mRNA-degrading activity was increased in protein extracts from YC-1-exposed aorta and was attenuated by pretreatment with actinomycin D and NS2028. Gelshift and supershift analyses using an adenylate-uridylate-rich ribonucleotide from the 3'-untranslated region of the alpha(1) mRNA and a monoclonal antibody directed against the mRNA-stabilizing protein HuR revealed HuR mRNA binding activity in aortic extracts, which was absent in extracts from YC-1-stimulated aortas. YC-1 decreased the expression of HuR, and this decrease was prevented by NS2028. Similarly, down-regulation of HuR by RNA interference in cultured rat aortic smooth muscle cells decreased alpha(1) mRNA and protein expression. We conclude that HuR protects the guanylyl cyclase alpha(1) mRNA by binding to the 3'-untranslated region. Activation of guanylyl cyclase decreases HuR expression, inducing a rapid degradation of guanylyl cyclase alpha(1) mRNA and lowering alpha(1) subunit expression as a negative feedback response.     

Allostery in recombinant soluble guanylyl cyclase from Manduca sexta

Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric full-length and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO off-rates for the approximately 100-kDa N-terminal heterodimeric fragment and increases the CO affinity by approximately 50-fold to 1.7 microm. Binding of NO leads to a transient six-coordinate intermediate, followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex (k(6-5) = 12.8 s(-1)). The conversion rate is insensitive to nucleotides, YC-1, and changes in NO concentration up to approximately 30 microm. NO release is biphasic in the absence of YC-1 (k(off1) = 0.10 s(-1) and k(off2) = 0.0015 s(-1)); binding of YC-1 eliminates the fast phase but has little effect on the slower phase. Our data are consistent with a model for allosteric activation in which sGC undergoes a simple switch between two conformations, with an open or a closed heme pocket, integrating the influence of numerous effectors to give the final catalytic rate. Importantly, YC-1 binding occurs in the N-terminal two-thirds of the protein. Homology modeling and mutagenesis experiments suggest the presence of an H-NOX domain in the alpha subunit with importance for heme binding.     

Role of conformational changes in the heme-dependent regulation of human soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is a receptor for endogenous and exogenous nitric oxide (NO) and is activated many fold upon its binding, making it a core enzyme in the nitric oxide signal transduction pathway. Much effort has been made to understand the link between binding of NO at the sGC heme and activation of the cyclase activity. We report here the first direct evidence for the role of conformational changes in transmitting the signal between the heme and cyclase domains. Using both circular dichroism (CD) and fluorescence spectroscopies, we have probed the effect that the sGC activators NO and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole (YC-1) and the inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) have on the structure of the protein. Surprisingly, binding of either ODQ or YC-1 to NO-bound sGC cause virtually identical changes in the far-UV CD spectra of sGC, reflecting a perturbation in the secondary structure of the enzyme. This change is absent upon binding of NO, YC-1 or ODQ alone. Using this and previous data, we propose a working model for the mechanism of activation of sGC by NO and YC-1 and inhibition by ODQ.     

BAY 41-2272 activates two isoforms of nitric oxide-sensitive guanylyl cyclase

Soluble guanylyl cyclase is an important target for endogenous nitric oxide and the guanylyl cyclase modulator, YC-1. Recently BAY 41-2272 was identified as a similar but more potent and more specific substance. While YC-1 also acts as non-specific phosphodiesterase inhibitor, BAY 41-2272 is devoid of an effect on phosphodiesterases. BAY 41-2272 has so far only been tested on the alpha(1)/beta(1) heterodimeric isoform of soluble guanylyl cyclase and its binding site has been mapped to a region in the alpha(1) subunit amino-terminal sequence. Although this region is poorly conserved in the alpha(2) subunit, we show in the current study that the alpha(2)/beta(1) heterodimeric enzyme isoform is activated by BAY 41-2272. Deletion analysis of the alpha(2) subunit and co-expression with the beta(1) subunit in the baculovirus/Sf9 system is consistent with the amino-terminal amino acids 104 to 401 of the alpha(2) subunit as binding site for BAY 41-2272.     

Dissociation of nitric oxide from soluble guanylate cyclase and heme-nitric oxide/oxygen binding domain constructs

Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length alpha1beta1 sGC, and the constructs beta1(1-194), beta1(1-385), and beta2(1-217), at 37 and 10 degrees C with and without the substrate analogue guanosine-5'-[(alpha,beta-methylene]triphosphate (GMPCPP) or the activator 3-(5'-hydroxymethyl-3'-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and beta2(1-217) at 37 degrees C and dramatically accelerated NO dissociation from sGC at 10 degrees C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 degrees C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity "closed" form, which releases NO slowly, and a higher activity "open" form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states.     

Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site

Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP, is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of binding of NO to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used (15)NO and (14)NO and anaerobic sequential mixing-freeze-quench electron paramagnetic resonance to unambiguously confirm that the heme Fe is the target of the second NO. The linear dependence on NO concentration up to 600 s(-1) for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e., the proximal site of the heme, but also supports a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of five-coordinate NO complexes with the ligand on either the distal or proximal site and supports the selective release of NO from the distal side of the transient bis-NO-sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC complex containing a proximal NO is formed after the binding of the second NO.     

YC-1 facilitates release of the proximal His residue in the NO and CO complexes of soluble guanylate cyclase

The benzylindazole compound YC-1 has been shown to activate soluble guanylate cyclase by increasing the sensitivity toward NO and CO. Here we report the action of YC-1 on the coordination of CO- and NO-hemes in the enzyme and correlate the events with the activation of enzyme catalysis. A single YC-1-binding site on the heterodimeric enzyme was identified by equilibrium dialysis. To explore the affect of YC-1 on the NO-heme coordination, the six-coordinate NO complex of the enzyme was stabilized by dibromodeuteroheme substitution. Using the dibromodeuteroheme enzyme, YC-1 converted the six-coordinate NO-heme to a five-coordinate NO-heme with a characteristic EPR signal that differed from that in the absence of YC-1. These results revealed that YC-1 facilitated cleavage of the proximal His-iron bond and caused geometrical distortion of the five-coordinate NO-heme. Resonance Raman studies demonstrated the presence of two iron-CO stretch modes at 488 and 521 cm(-1) specific to the YC-1-bound CO complex of the native enzyme. Together with the infrared C-O stretching measurements, we assigned the 488-cm(-1) band to the iron-CO stretch of a six-coordinate CO-heme and the 521-cm(-1) band to the iron-CO stretch of a five-coordinate CO-heme. These results indicate that YC-1 stimulates enzyme activity by weakening or cleaving the proximal His-iron bond in the CO complex as well as the NO complex.     

Nitric oxide. Potentiation of NO-dependent activation of soluble guanylate cyclase: (Patho)physiological and pharmacotherapeutic importance

The review highlights the molecular mechanism underlying the physiological effects of nitric oxide (NO), the role of signaling system: NO-soluble guanylate cyclase-cyclic 3′,5′-guanosine monophosphate (cGMP) in the realization of NO action. This review considers data on basic chemical characteristics of guanylate cyclase, such as the subunits structure, isoforms, modern concepts of the catalytic and regulatory centers of this enzyme. Realization of physiological effects of NO by guanylate cyclase depends on its heme prostetic group. NO-dependent activation of guanylate cyclase may be synergistically increased by a new NO-independent, allosteric activator of soluble guanylate cyclase-YC-1-(benzyl indasol derivative). Special attention is paid to the data on guanylate cyclase sites responcible for binding of the enzyme with YC-1 and the possible molecular mechanism underlying the synergistic increase of NO-dependent activation of soluble guanylate cyclase by YC-1. New compounds of endogenous nature capable to potentiate and synergistically increase the activation of guanylate cyclase by NO-donors have been found and investigated. The important physiological, pharmacotherapeutical and pathophysiological significance of this new fact is discussed.     

Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line.

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.