sGC Subunits Research Articles

The process of S-nitrosation in sGC β1(1–194) revealed by infrared spectroscopy

Soluble guanylate cyclase (sGC) is the most important receptor for the signaling molecule NO. NO activates sGC by binding to its heme cofactor and reacts with free thiols in the protein itself. The S-nitrosation of cysteine thiols affects the activity of soluble guanylate cyclase (sGC). In this study, infrared (IR) spectroscopy and site-directed mutagenesis were used to investigate S–H vibration and the process of S-nitrosation in the β1 subunit (amino acids 1–194) of sGC. Fourier transform IR spectroscopy revealed that wild-type and mutants (C78S and C122S) of sGC β1(1–194) exhibited S–H peaks around 2560cm−1. The signals were attenuated in the IR spectra of S-nitrosoglutathione-treated mutants, demonstrating that S-nitrosation in sGC β1(1–194) occured at residues C78 and C122, and the process of the reaction was GSNO concentration-dependent.

Prolonged therapy with the soluble guanylyl cyclase activator BAY 60-2770 restores the erectile function in obese mice.

Cardiovascular and endocrine-metabolic diseases associated with increased oxidative stress such as obesity lead to erectile dysfunction (ED). Activators of soluble guanylyl cyclase (sGC) such as BAY 60-2770 reactivate the heme-oxidized sGC in vascular diseases. This study aimed to evaluate the effects of 2-week oral intake with BAY 60-2270 on a murine model of obesity-associated ED. C57BL/6 male mice were fed for 12 weeks with standard chow or high-fat diet. Lean and obese mice were treated with BAY 60-2770 (1 mg/kg/day, 2 weeks). Measurements of intracavernosal pressure (ICP), along with acetylcholine (10(-9) to 10(-5) M) and electrical field stimulation (EFS; 4-10 Hz)-induced corpus cavernosum relaxations in vitro, were obtained. Levels of cyclic guanosine monophosphate (cGMP), reactive oxygen species (ROS), and sGC protein expressions in cavernosal tissues were measured. Cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly lower in obese compared with lean mice (P < 0.05). Two-week therapy with BAY 60-2770 fully reversed the decreased ICP in obese group. Acetylcholine-induced cavernosal relaxations were 45% lower (P < 0.001) in obese mice, which were fully restored by BAY 60-2770 treatment. Likewise, the EFS-induced relaxations in obese mice were restored by BAY 60-2770. Basal cGMP content in erectile tissue was 68% lower (P < 0.05) in obese mice, an effect normalized by BAY 60-2770. Levels of ROS were 52% higher (P < 0.05) whereas protein expression of α1 sGC subunit was reduced in cavernosal tissue of obese mice, both of which were normalized by BAY 60-2770. In lean group, BAY 60-2770 did not significantly affect any functional, biochemical, or molecular parameter analyzed. Two-week therapy with BAY 60-2770 restores the erectile function in obese mice that is associated with reduced ROS levels, up-regulation of α1 sGC subunit, and increased cGMP levels in the erectile tissue.

Influence of cinaciguat on gastrointestinal motility in apo-sGC mice.

Cinaciguat (BAY 58-2667), an NO- and heme-independent sGC activator, was shown to be more effective when the heme-group of sGC is oxidized in vascular tissue. In apo-sGC mice (sGCβ1 (His105Phe) knockin) both sGC isoforms (sGCα1 β1 and sGCα2 β1 ) are heme-deficient and can no longer be activated by NO; these mice, showing decreased gastrointestinal nitrergic relaxation and decreased gastric emptying, can be considered as a model to study the consequence of heme-oxidation in sGC. Our aim was to compare the influence of cinaciguat, on in vitro muscle tone of gastrointestinal tissues, and on gastric emptying in WT and apo-sGC mice. Gastrointestinal smooth muscle strips were mounted in organ baths for isometric force recording and cGMP levels were determined by enzyme immunoassay. Protein levels of sGC subunits were assessed by immunoblotting. Gastric emptying was determined by phenol red recovery. Although protein levels of the sGC subunits were lower in gastrointestinal tissues of apo-sGC mice, cinaciguat induced concentration-dependent relaxations and increased cGMP levels in apo-sGC fundus and colon to a similar or greater extent than in WT mice. The sGC inhibitor ODQ increased cinaciguat-induced relaxations and cGMP levels in WT fundus and colon. In apo-sGC antrum, pylorus and jejunum, cinaciguat was not able to induce relaxations. Cinaciguat did not improve delayed gastric emptying in apo-sGC mice. Cinaciguat relaxes the fundus and colon efficiently when sGC is in the heme-free condition; the non-effect of cinaciguat in pylorus explains its inability to improve the delayed gastric emptying in apo-sGC mice.

What is the role of S‐nitrosation of soluble guanylyl cyclase in the heart? (698.9)

Nitric oxide (NO) is involved in cardioprotection against ischemia/reperfusion (I/R) injury. Soluble guanylyl cyclase (sGC) produces cGMP when it is stimulated by NO. Association of the α and β subunit of sGC is required for cGMP‐forming activity. NO protects the heart from I/R via a cGMP‐dependent and a S‐nitrosation (SNO)‐dependent pathway. Preliminary evidence showed that sGC‐overexpression increases S‐nitrosation of specific proteins (SNO‐proteins) in rat neonatal cardiomyocytes. These sGC‐dependent SNO‐proteins were identified by Mass Spectrometry and are mostly associated with intercalated disc and cytoskeleton, including integrin‐linked kinase (ILK) and desmin. sGC is heavily S‐nitrosated in the presence of nitrosating agent. Thus, we hypothesize that sGC acts as a transnitrosylase in the heart, e.g. transfers its SNO to specific proteins. Intriguingly, sGC‐α and β subunits localize differently in cardiomyocytes. β is at the nuclear envelope and sarcolemma, while α is in cytosol, suggesting that each subunit might have a separate function, independent of cGMP‐forming activity. In cardiomyocytes treated with a nitrosating agent, overexpressing a single sGC subunit, either α or β, was sufficient to increase SNO level of ILK and desmin. Hence, we propose that sGC could modulate cardioprotection by increasing protein‐SNO, in addition to the NO‐cGMP dependent pathway.Grant Funding Source: Supported by 6R01 GM067640

Cell-specific expression and immunolocalization of nitric oxide synthase isoforms and soluble guanylyl cyclase α and β subunits in postnatal porcine uteri

The aim of the present study was to investigate the cellular expression and immunolocalization of nitric oxide synthase (NOS) isoforms and soluble guanylyl cyclase (sGC) subunits in postnatal porcine uteri. Immunohistochemical experiments showed that three isoforms of NOS were mainly localized in the uterine luminal and glandular epithelium and myometrium, and the intensity of immunostaining for iNOS and eNOS was increased gradually with temporal development of the postnatal uterus. In addition, sGC subunits, sGCα1 and β, were present in the uterine luminal and glandular epithelium, myometrium and stromal cells. The uterine NOS activity data showed that the total NOS and iNOS activities were significantly increased at postnatal days 21 and 35. Although constitutive NOS activity was increased at postnatal day 21, it decreased subsequently at postnatal day 35. Immunoblot analysis revealed that iNOS protein expression was significantly increased at postnatal days 21 and 35. Furthermore, sGCα1 protein expression was not significantly changed throughout days 7 to 35. Collectively, our findings suggest that NO/cGMP signaling is involved in the process of postnatal porcine uterine development.

Notch activation augments nitric oxide/soluble guanylyl cyclase signaling in immortalized ovarian surface epithelial cells and ovarian cancer cells

Nitric oxide (NO) is generated by tumor, stromal and endothelial cells and plays a multifaceted role in tumor biology. Many physiological functions of NO are mediated by soluble guanylyl cyclase (sGC) and NO/sGC signaling has been shown to promote proliferation and survival of ovarian cancer cells. However, how NO/sGC signaling is modulated in ovarian cancer cells has not been studied. The evolutionarily conserved Notch signaling pathway plays an oncogenic role in ovarian cancer. Here, we report that all three ovarian cancer cell lines we examined express a higher level of GUCY1B3 (the β subunit of sGC) compared to non-cancerous immortalized ovarian surface epithelial (IOSE) cell lines. Interestingly, the highest expression of GUCY1B3 in ovarian cancer OVCAR3 cells is concurrent with the expression of Notch3. In IOSE cells, forced activation of Notch3 increases the expression of GUCY1B3, NO-induced cGMP production, and the expression of cGMP-dependent protein kinase (PKG), thereby enhancing NO- and cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP, a direct PKG substrate protein). In contrast, inhibition of Notch by DAPT reduces GUCY1B3 expression and NO-induced cGMP production and VASP phosphorylation in OVCAR3 cells. Finally, we confirmed that inhibition of sGC by ODQ decreases growth of ovarian cancer cells. Together, our work demonstrates that Notch is a positive regulator of NO/sGC signaling in IOSE and ovarian cancer cells, providing the first evidence that Notch and NO signaling pathways interact in IOSE and ovarian cancer cells.

Volume overload induces differential spatiotemporal regulation of myocardial soluble guanylyl cyclase in eccentric hypertrophy and heart failure

Nitric oxide activation of soluble guanylyl cyclase (sGC) blunts the cardiac stress response, including cardiomyocyte hypertrophy. In the concentric hypertrophied heart, oxidation and re-localization of myocardial sGC diminish cyclase activity, thus aggravating depressed nitric oxide–cyclic guanosine monophosphate (NO–cGMP) signaling in the pressure-overloaded failing heart. Here, we hypothesized that volume-overload differentially disrupts myocardial sGC activity during early compensated and late decompensated stages of eccentric hypertrophy. To this end, we studied the expression, redox state, subcellular localization, and activity of sGC in the left ventricle of dogs subjected to chordal rupture-induced mitral regurgitation (MR). Unoperated dogs were used as Controls. Animals were studied at 4weeks and 12months post chordal rupture, corresponding with early (4wkMR) and late stages (12moMR) of eccentric hypertrophy. We found that the sGC heterodimer subunits relocalized away from caveolae-enriched lipid raft microdomains at different stages; sGCβ1 at 4wkMR, followed by sGCα1 at 12moMR. Moreover, expression of both sGC subunits fell at 12moMR. Using the heme-dependent NO donor DEA/NO and NO-/heme-independent sGC activator BAY 60-2770, we determined the redox state and inducible activity of sGC in the myocardium, within caveolae and non-lipid raft microdomains. sGC was oxidized in non-lipid raft microdomains at 4wkMR and 12moMR. While overall DEA/NO-responsiveness remained intact in MR hearts, DEA/NO responsiveness of sGC in non-lipid raft microdomains was depressed at 12moMR. Caveolae-localization protected sGC against oxidation. Further studies revealed that these modifications of sGC were also reflected in caveolae-localized cGMP-dependent protein kinase (PKG) and MAPK signaling. In MR hearts, PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) disappeared from caveolae whereas caveolae-localization of phosphorylated ERK5 increased. These findings show that differential oxidation, re-localization, and expression of sGC subunits distinguish eccentric from concentric hypertrophy as well as compensated from decompensated heart failure.

Activation of Haem-Oxidized Soluble Guanylyl Cyclase with BAY 60-2770 in Human Platelets Lead to Overstimulation of the Cyclic GMP Signaling Pathway

Background and AimsNitric oxide-independent soluble guanylyl cyclase (sGC) activators reactivate the haem-oxidized enzyme in vascular diseases. This study was undertaken to investigate the anti-platelet mechanisms of the haem-independent sGC activator BAY 60-2770 in human washed platelets. The hypothesis that sGC oxidation potentiates the anti-platelet activities of BAY 60-2770 has been tested.MethodsHuman washed platelet aggregation and adhesion assays, as well as flow cytometry for αIIbβ3 integrin activation and Western blot for α1 and β1 sGC subunits were performed. Intracellular calcium levels were monitored in platelets loaded with a fluorogenic calcium-binding dye (FluoForte).ResultsBAY 60-2770 (0.001–10 µM) produced significant inhibition of collagen (2 µg/ml)- and thrombin (0.1 U/ml)-induced platelet aggregation that was markedly potentiated by the sGC inhibitor ODQ (10 µM). In fibrinogen-coated plates, BAY 60-2770 significantly inhibited platelet adhesion, an effect potentiated by ODQ. BAY 60-2770 increased the cGMP levels and reduced the intracellular Ca2+ levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 µM) inhibited platelet aggregation and Ca2+ levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen- and thrombin-induced αIIbβ3 activation was markedly inhibited by BAY 60-2770 that was further inhibited by ODQ. The effects of sodium nitroprusside (3 µM) were all prevented by ODQ. Incubation with ODQ (10 µM) significantly reduced the protein levels of α1 and β1 sGC subunits, which were prevented by BAY 60-2770.ConclusionThe inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca2+ levels and αIIbβ3 activation are all potentiated in haem-oxidizing conditions. BAY 60-2770 prevents ODQ-induced decrease in sGC protein levels. BAY 60-2770 could be of therapeutic interest in cardiovascular diseases associated with thrombotic complications.

The G-protein regulator LGN modulates the activity of the NO receptor soluble guanylate cyclase

sGC (soluble guanylate cyclase) is the main mediator of NO signalling. Biochemical and physiological studies suggest that, besides NO, in vivo regulation of sGC involves direct interaction with other proteins. Using yeast two-hybrid screening, we identified that the multidomain LGN (Leu-Gly-Asn repeat-enriched protein) interacts with both α1 and β1 sGC subunits. LGN and sGC co-localized in the cell cytoplasm, and the LGN-sGC complex was co-immunoprecipitated from cells expressing both proteins and from native tissues. Their interaction requires the N-terminal tetratricopeptide repeats of LGN, but does not require the N-terminal portions of α1 or β1 sGC subunits. Overexpression of LGN decreases the activity of cellular sGC, whereas knockdown of LGN mRNA and protein correlated with increased sGC activity. Although purified LGN interacts directly with purified sGC, the inhibitory effect in vitro is observed only after supplementation of cell lysate to the reaction. Although resting sGC and sGC activated by the stimulator BAY41-2272 have very similar LGN-IC50 values to the NO-stimulated sGC, they have a much higher Hill coefficient, suggesting co-operative binding with respect to LGN in the low-activated state of sGC. AGS3 (activator of G-protein signalling 3), the closest LGN homologue, also inhibits sGC. The interaction of sGC with these scaffolding proteins may expand the cross-talk between NO/cGMP signalling and other cellular pathways and tailor sGC function to specific tissues or signals.

Abstract 178: Volume Overload Induced by Mitral Regurgitation Alters Spatial Regulation of Nitric Oxide/Reactive Nitrogen Species Signaling

Volume-overload (VO), as seen in regurgitant valvular disease, large myocardial infarction, and severe cardiac systolic dysfunction, triggers eccentric hypertrophy. Given that nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) modulate cardiomyocyte hypertrophy, apoptosis, and cardioprotection, differential regulation of these signals may distinguish eccentric from the more commonly studied concentric hypertrophy. We recently showed that pressure-overload (PO) induces relocalization and oxidation of the NO receptor soluble guanylyl cyclase (sGC), thereby diminishing cyclase activity and cGMP cardioprotection. The effects of volume-overload on NO and cGMP signaling are unknown. We tested the hypothesis that VO induces relocalization but not oxidation of sGC, thereby disrupting spatial regulation of NO-cGMP signaling without depressing cyclase activity. Volume-overload was established by chordal rupture-induced mitral regurgitation in dogs. We compared intracellular localization and activity of sGC in VO and control LVs (N=5 per group). Both sGC subunits were detected within and outside of caveolae-enriched lipid raft microdomains (Cav3+LR). In VO hearts, sGCβ1 fell in expression by nearly 50% and relocalized away from Cav3+LR to non-lipid raft microdomains (NLR). Despite VO-induced sGCβ1 changes, overall NO-stimulated sGC activity was preserved. An enhanced response to heme/NO-independent sGC activator BAY 60-2770 suggested that a subset of sGC was heme-oxidized in VO hearts, though to a much lesser degree than in PO hearts. As in PO hearts, Cav3+LR appear to protect sGC from heme-oxidation in VO hearts. Initial study of downstream reactive nitrogen species (RNS) and cGMP signaling supported our theory that VO altered spatial regulation of NO-cGMP signaling. Also, a trend towards increased overall tyrosine-nitration, predominantly within NLR, was observed in VO hearts. Volume-overload shifted cardiac NO-cGMP signaling from Cav3+LR to NLR microdomains without depressing NO/heme-dependent sGC activation. These findings suggest that differential spatial regulation of NO/RNS signaling, rather than simply increased RNS signaling, might drive the distinct molecular pathophysiology of eccentric hypertrophy.

Hydrogen Peroxide Alters Splicing of Soluble Guanylyl Cyclase and Selectively Modulates Expression of Splicing Regulators in Human Cancer Cells

BackgroundSoluble guanylyl cyclase (sGC) plays a central role in nitric oxide (NO)-mediated signal transduction in the cardiovascular, nervous and gastrointestinal systems. Alternative RNA splicing has emerged as a potential mechanism to modulate sGC expression and activity. C-α1 sGC is an alternative splice form that is resistant to oxidation-induced protein degradation and demonstrates preferential subcellular distribution to the oxidized environment of endoplasmic reticulum (ER).Methodology/Principal FindingsHere we report that splicing of C-α1 sGC can be modulated by H2O2 treatment in BE2 neuroblastoma and MDA-MD-468 adenocarcinoma human cells. In addition, we show that the H2O2 treatment of MDA-MD-468 cells selectively decreases protein levels of PTBP1 and hnRNP A2/B1 splice factors identified as potential α1 gene splicing regulators by in silico analysis. We further demonstrate that down-regulation of PTBP1 by H2O2 occurs at the protein level with variable regulation observed in different breast cancer cells.Conclusions/SignificanceOur data demonstrate that H2O2 regulates RNA splicing to induce expression of the oxidation-resistant C-α1 sGC subunit. We also report that H2O2 treatment selectively alters the expression of key splicing regulators. This process might play an important role in regulation of cellular adaptation to conditions of oxidative stress.

The roles of cysteines in the heme domain of human soluble guanylate cyclase

Abstract Soluble guanylate cyclase (sGC) is a critical heme-containing enzyme involved in NO signaling. The dimerization of sGC subunits is necessary for its bioactivity and its mechanism is a striking and an indistinct issue. The roles of heme domain cysteines of the sGC on the dimerization and heme binding were investigated herein. The site-directed mutations of three conserved cysteines (C78A, C122A and C174S) were studied systematically and the three mutants were characterized by gel filtration analysis, UV–vis spectroscopy and heme transfer examination. Cys78 was involved in heme binding but not referred to the dimerization, while Cys174 was demonstrated to be involved in the homodimerization. These results provide new insights into the cysteine-related dimerization regulation of sGC.

Cell-specific expression and immunolocalization of nitric oxide synthase isoforms and soluble guanylyl cyclase α1 and β1 subunits in the ovary of fetal, neonatal and immature pigs

The present study is designed to investigate the cellular expression and immunolocalization of three different nitric oxide synthase (NOS) isoforms and soluble guanylyl cyclase (sGC) subunits in the porcine ovary. Our results showed that in the fetal and neonatal pigs, all three isoforms of NOS were mainly localized in the oocyte and showed the expression of gradual increase in the granulosa cell and theca cell with the growing follicle. In addition, subunits of the sGC, sGC α1 and β1 were mainly expressed in the granulosa cell in precious studies. The bioactivity of total NOS, eNOS, iNOS and nNOS was detected in the ovary and were higher at prenatal stages compared to postnatal stages. However, the activities of nNOS were no different between prenatal stages and postnatal stages. Taken together, our findings suggested that the NOS/sGC pathway may be involved in the follicular formation and development in the porcine ovary.

Hypoxia induces downregulation of soluble guanylyl cyclase β1 by miR-34c-5p

Soluble guanylyl cyclase (sGC) is the principal receptor for nitric oxide (NO) and crucial for the control of various physiological functions. The β1 subunit of sGC is obligatory for the biological stability and activity of the sGC heterodimer. MicroRNAs (miRNAs) are important regulators of gene expression and exert great influences on diverse biological activities. The aim of the present study was to determine whether or not the expression of sGCβ1 is specifically regulated by miRNAs. We report that miR-34c-5p directly targets sGCβ1 under hypoxia. Bioinformatics analysis of the sGCβ1 3'-untranslated region (3'-UTR) revealed a putative binding site for miR-34b-5p and miR-34c-5p, but only miR-34c-5p inhibited luciferase activity through interaction with sGCβ1 3'-UTR in HEK293T cells. Site-directed mutagenesis of the putative miR-34c-5p binding site abolished the negative regulation of luciferase expression. Overexpression of miR-34c-5p repressed the expression of sGCβ1 in stable cell lines, which was reversed by miR-34c-5p-specific sponge. Inoculation of mouse lung tissues in vitro with lentivirus bearing miR-34c-5p significantly decreased both the expression of sGCβ1 and NO-stimulated sGC activity, which was also rescued by miR-34c-5p-specific sponge. Furthermore, we identified the putative Sp1-binding site in the promoter region of miR-34c-5p. Luciferase reporter constructs revealed that Sp1 directly binds to the wild-type promoter of miR-34c-5p, which was confirmed by chromatin immunoprecipitation. In summary, these findings reveal that miR-34c-5p directly regulates sGCβ1 expression, and they identify the key transcription factor Sp1 that governs miR-34c-5p expression during hypoxia.

Nitric Oxide-Sensitive Guanylyl Cyclase Is Differentially Regulated by Nuclear and Non-Nuclear Estrogen Pathways in Anterior Pituitary Gland

17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway.

Identification of Residues in the Heme Domain of Soluble Guanylyl Cyclase that are Important for Basal and Stimulated Catalytic Activity

Nitric oxide signals through activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain) to the effector domain (catalytic domain), in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105) of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.

Study on the involvement of soluble guanylyl cyclase and its different isoforms in carbon monoxide and carbon monoxide releasing molecule-2 induced vasodilatation

Besides nitric oxide, carbon monoxide (CO) also activates soluble guanylyl cyclase (sGC). CO as well as the CO-donor CORM-2 have been shown to possess vasodilatory properties. Whether these vasodilatory properties by CO can be attributed to sGC activation is still a matter of debate. The aim of this study was to examine the involvement of sGC and its different subunits in CO and CORM-2 induced vasodilatation within different vascular tissues. Isometric tension recordings were performed using mice isolated aortic rings, femoral artery ring segments as well as corpora cavernosa (CC). To be able to distinguish between the different sGC subunits we evaluated responses to saturated CO solutions and CORM-2 in both sGCa1-/- and sGCβ1KI/KI mice and their wild-type controls. Saturated CO solution was unable to relax mice isolated blood vessels, whereas it induced concentration-dependent relaxations in mice CC. In CC of wild-type mice, the response to CO was completely inhibited by the sGC inhibitor ODQ. The involvement of sGC in the CO-induced corporal relaxation was further confirmed by the loss of response to CO in CC isolated from sGCβ1KI/KI mice. Moreover, the vasodilatory responses of CO in the corporal tissue of sGCa1-/- mice were strongly inhibited although not completely abolished. In contrast to CO, CORM-2 was able to relax all vascular tissues examined in the present study, although ODQ only partially blocked the response to CORM-2 in the aorta. Interestingly ODQ did not affect the CORM-2 induced relaxation in the femoral arteries and the CC, indicating that sGC is not involved, which was confirmed using the transgenic mice. This study clearly illustrates that the molecular mechanism of CORM-2 induced vasorelaxation differs from that of CO induced vasorelaxation. While the CO induced vasorelaxation depends on activation of sGC, primarily the sGCa1β1 heterodimer, the vasorelaxing properties of CORM-2 are only partially dependent or even completely independent upon sGC activation. The observation that CO is more effective in relaxing CC tissues than other cardiovascular tissues investigated in the present study suggests that the heme-oxygenase/CO pathway may present a potential new target for therapeutic approaches towards erectile dysfunction.

A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase

Human soluble guanylate cyclase (sGC), a critical heme-containing enzyme in the NO-signaling pathway of eukaryotes, is an αβ heterodimeric hemoprotein. Upon the binding of NO to the heme, sGC catalyzes the conversion of GTP to cyclic GMP, playing a crucial role in many physiological processes. However, the specific contribution of the α and β subunits of sGC in the intact heme binding remained intangible. The recombinant human sGC α1 subunit has been expressed in Escherichia coli and characterized for the first time. The heme binding and related NO/CO binding properties of both the α1 subunit and the β1 subunit were investigated via heme reconstitution, UV-vis spectroscopy, EPR spectroscopy, stopped-flow kinetics, and homology modeling. These results indicated that the α1 subunit of human sGC, lacking the conserved axial ligand, is likely to interact with heme noncovalently. On the basis of the equilibrium and kinetics of CO binding to sGC, one possible CO binding model was proposed. CO binds to human sGCβ195 by simple one-step binding, whereas CO binds to human sGCα259, possibly from both axial positions through a more complex process. The kinetics of NO dissociation from human sGC indicated that the NO dissociation from sGC was complex, with at least two release phases, and human sGCα259 has a smaller k (1) but a larger k (2). Additionally, the role of the cavity of the α1 subunit of human sGC was explored, and the results indicate that the cavity likely accommodates heme. These results are beneficial for understanding the overall structure of the heme binding site of the human sGC and the NO/CO signaling mechanism.

Aspartate 102 in the Heme Domain of Soluble Guanylyl Cyclase Has a Key Role in NO Activation

Nitric oxide (NO) is involved in the physiology and pathophysiology of the cardiovascular and neuronal systems via activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. Recent structural studies have allowed a better understanding of the residues that dictate the affinity and binding of NO to the heme and the resulting breakage of the bond between the heme iron and histidine 105 (H105) of the β subunit of sGC. Still, it is unknown how the breakage of the iron-His bond translates into NO-dependent increased catalysis. Structural studies on homologous H-NOX domains in various states pointed to a role for movement of the H105 containing αF helix. Our modeling of the heme-binding domain highlighted conserved residues in the vicinity of H105 that could potentially regulate the extent to which the αF helix shifts and/or propagate the activation signal once the covalent bond with H105 has been broken. These include a direct interaction of αF helix residue aspartate 102 (D102) with the backbone nitrogen of F120. Mutational analysis of this region points to an essential role of the interactions in the vicinity of H105 for heme stability and identifies D102 as having a key role in NO activation following breakage of the iron-His bond.

G‐protein regulator LGN inhibits the activity of nitric oxide receptor soluble guanylyl cyclase

Soluble guanylyl cyclase (sGC) is a heme‐containing enzyme which converts GTP to cGMP. SGC is referred to as nitric oxide (NO) receptor, as it is strongly stimulated by NO. Although the mechanism of sGC activation by NO received a lot of attention, the processes of sGC deactivation are less understood. In this report we demonstrate inhibition of sGC by a LGN‐dependent mechanism. LGN is a known regulator of Gα proteins. We identified LGN as a protein interacting with both α1 and β1 sGC subunits by yeast two‐hybrid screening and by co‐immunoprecipitation of sGC and LGN from BE2 human neuroblastoma cell line. Transient expression of LGN in MDA468 breast cancer cells markedly decreased sGC activity in a gene‐dose dependent fashion. When the effect of purified LGN protein on the activity of purified sGC was tested in vitro, we found no evidence of inhibition, suggesting that LGN does not directly affect sGC function. However, when the same experiment was performed in the lysate of COS7 cell, we observed a marked inhibition of sGC. These data suggest that LGN may function as an adaptor protein for additional cellular factors inhibiting sGC. Since LGN is known to regulate G‐proteins by association with Gα subunit, it may function as an adaptor protein for a complex consisting of sGC, LGN and Gα, thus connecting the NO‐dependent and G‐protein signaling pathways.