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.     

YC-1-like potentiation of nitric oxide-dependent activation of soluble guanylate cyclase by adrenochrom

The influence of adrenochrome and YC-1 activation of human platelet soluble guanylate cyclase was investigated. Adrenochrome (0.1–10.0 μM) had no effect on the basal activity, but it potentiated in a concentration- dependent manner the spermine NONO-induced activation of this enzyme. Adrenochrome also sensitized guanylate towards nitric oxide (NO) and produced the leftward shift of the spermine NONO concentration response curve. Addition of adrenochrome decreased the YC-1-induced leftward shift of the spermine NONO concentration response curve. Adrenochrome also inhibited enzyme activation byYC-1. Thus, synergistic activation of NO-stimulated guanylate cyclase activity by adrenochrome represents a new biochemical effect of this compound and indicates that adrenochrome may act as an endogenous regulator of the NO-dependent stimulation of soluble guanylate cyclase. This new property of adrenochrome, similar to YC-1 but more effective, should be taken into consideration especially under conditions of adrenochrome overproduction in the body.     

In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum

Carbon monoxide induces delayed neurological and neuropathological alterations, including memory loss and cognitive impairment. The bases for the delay remain unknown. Activation of soluble guanylate cyclase by nitric oxide modulates some forms of learning and memory. Carbon monoxide binds to soluble guanylate cyclase, activating it but interfering with its activation by nitric oxide. The aim of this work was to assess whether exposure of rats to carbon monoxide alters the activity of soluble guanylate cyclase or its modulation by nitric oxide in cerebellum or cerebral cortex. Rats exposed chronically or acutely to carbon monoxide were killed 24 h or 7 days later. Acute carbon monoxide exposure decreased cyclic guanosine monophosphate (cGMP) content and reduced activation of soluble guanylate cyclase by nitric oxide. Cortex was more sensitive than cerebellum to chronic exposure, which reduced activation of soluble guanylate cyclase by nitric oxide in cortex. In cerebellum, chronic exposure induced delayed impairment of soluble guanylate cyclase activation by nitric oxide. Acute exposure effects were also stronger at 7 days than at 24 h after exposure. This delayed impaired modulation of soluble guanylate cyclase by nitric oxide may contribute to delayed memory loss and cognitive impairment in humans exposed to carbon monoxide.     

Acidic triazoles as soluble guanylate cyclase stimulators

A series of acidic triazoles with activity as soluble guanylate cyclase stimulators is described. Incorporation of the CF(3) triazole improved the overall physicochemical and drug-like properties of the molecule and is exemplified by compound 25.     

Evolution of the membrane guanylate cyclase transduction system

Almost four decades of research in the field of membrane guanylate cyclases is discussed in this review. Primarily, it focuses on the chronological development of the field, recognizes major contributions of the original investigators, corrects certain misplaced facts, and projects its future trend.     

Potentiation of NO-dependent activation of soluble guanylate cyclase by 5-nitroisatin and the antiviral agent arbidol

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. The effect of isatin derivatives, 5-nitroisatin and arbidol (an antiviral agent) on spermine NONO-induced activation of human platelet soluble guanylate cyclase has been investigated. 5-Nitroisatin and arbidol had no effect on basal activity, but synergistically increased in a concentration-dependent manner the spermine NONO-induced activation of this enzyme. 5-Nitroisatin and arbidol, like YC-1, sensitized guanylate cyclase towards nitric oxide (NO) and produced a leftward shift of the spermine NONO concentration response curve. However, both compounds did not influence the activation of guanylate cyclase by YC-1 and did not change the synergistic increase of spermine NONO-induced activation of soluble guanylate cyclase in the presence of YC-1. This suggests that 5-nitroisanin and arbidol did not compete with YC-1. Addition of isatin did not change the synergistic increase in the spermine NONO-induced guanylate cyclase activation by 5-nitroisatin and arbidol and did not influence a leftward shift of the spermine NONO concentration response curve produced by these compounds. These data suggest lack of competitive interaction between isatin and both its derivatives used.     

Heat shock protein 90 regulates stabilization rather than activation of soluble guanylate cyclase

Endothelium-derived nitric oxide (NO) activates the heterodimeric heme protein soluble guanylate cyclase (sGC) to form cGMP. In different disease states, sGC levels and activity are diminished possibly involving the sGC binding chaperone, heat shock protein 90 (hsp90). Here we show that prolonged hsp90 inhibition in different cell types reduces protein levels of both sGC subunits by about half, an effect that was prevented by the proteasome inhibitor MG132. Conversely, acute hsp90 inhibition affected neither basal nor NO-stimulated sGC activity. Thus, hsp90 is a molecular stabilizer for sGC tonically preventing proteasomal degradation rather than having a role in short-term activity regulation.     

Identification of residues crucially involved in soluble guanylate cyclase activation

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.     

ATP binding is required for physiological activation of retinal guanylate cyclase

ATP bound to retinal guanylate cyclase (retGC)/membranes prior to the assay (pre-binding effect) and during the assay (direct effect) further enhances retGC activity stimulated by GC-activating proteins (GCAPs). Here we investigate differences between these two effects. We found that the pre-binding effect, but not the direct effect, was absent in membranes pre-washed with Mg(2+)-free hypotonic buffers, that the pre-binding effect, but not the direct effect, was strictly limited to GCAP-stimulated retGC activity, and that these two effects were independent and additive rather than being synergistic. Pre-incubation with amiloride enhanced GCAP2-activated retGC activity in a manner similar to that by ATP pre-binding; however, amiloride did not directly stimulate the retGC activity. These results indicate that these two effects are mechanistically different. Levels of retGC activation by these effects and conditions required for these effects indicate that only the mechanism involving ATP pre-binding is physiologically relevant to retGC activation.     

Caspase-6 mediated cleavage of guanylate cyclase alpha 1 during deoxycholate-induced apoptosis: Protective role of the nitric oxide signaling module

Hydrophobic bile acids such as deoxycholate are known tumor promoters in the gastrointestinal tract. We have previously shown that deoxycholate induces apoptosis in colon epithelial cells and that these cells can be made resistant to deoxycholate-induced apoptosis. We now show that the nitric oxide synthase/nitric oxide/guanylate cyclase/cyclic guanosine monophosphate/cGMP-activated protein kinase (NOS/NO/GC/cGMP/PKG) signaling module contributes, in part, to the observed resistance of the cultured DOC-resistant colon epithelial cells (HCT-116R) using pharmacological inhibitors/antagonists (NS2028, Rp-8pCPT-cGMP, KT5823) of members of this signaling module. A novel finding from this study is the caspase-6 mediated cleavage of guanylate cyclase alpha 1 during deoxycholate-induced apoptosis of deoxycholate-sensitive HCT-116SA cells and the absence of guanylate cyclase alpha 1 cleavage in deoxycholate-treated HCT-116R resistant cells using Western blot analyses. This cleavage was specific to caspases as lysosomal, proteasomal, serine protease, cathepsin and calpain inhibitors failed to prevent the cleavage, whereas a general caspase inhibitor and a specific caspase-6 inhibitor did prevent guanylate cyclase alpha 1 cleavage.     

Surface plasmon resonance using the catalytic domain of soluble guanylate cyclase allows the detection of enzyme activators

Soluble Guanylate Cyclase (sGC) is the receptor for the signalling agent nitric oxide (NO) and catalyses the production of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP). The enzyme is an attractive drug target for small molecules that act in the cardiovascular and pulmonary systems, and has also shown to be a potential target in neurological disorders. We have discovered that 5-(indazol-3-yl)-1,2,4-oxadiazoles activate the enzyme in the absence of added NO and shown they bind to the catalytic domain of the enzyme after development of a surface plasmon resonance assay that allows the biophysical detection of intrinsic binding of ligands to the full length sGC and to a construct of the catalytic domain.     

Oxygen concentration regulates NO-dependent relaxation of aortic smooth muscles

Nitric oxide (NO) functions as an endothelium-derived relaxation factor and regulates vascular resistance. Recent studies in this laboratory (Arch. Biochem. Biophys. 323, 27–32, 1995) revealed that the lifetime of NO significantly increased at physiologically low levels of oxygen concentrations and, hence, this gaseous radical strongly inhibited mitochondrial electron transport for a fairly long duration at low oxygen concentrations. The present work describes the effect of oxygen concentration on NO-induced relaxation and guanylate cyclase (GC) activity of endothelium-denuded aorta of the rat. Both NO and 2,2′-hydroxynitrosohydrazono)bis-ethanamine (NOC18), an NO donor, induced the relaxa-tion of endothelium-denuded helical segments of rat aorta which were contracted by norepinephrine. NO-dependent relaxation of arterial specimens was enhanced by lowering oxygen concentration in the medium with concomitant increase in their cGMP levels. Anoxia induced the relaxation of the aorta by some NO-enhanceable and methylene blue-insensitive mechanism. These results suggested that local concentrations of oxygen might play important roles in the regulation of NO-dependent GC activity and vascular tonus of resistance arteries.     

Inhibition of NO-dependent soluble human platelet guanylate cyclase by isatin

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. Physiologically relevant concentrations of isatin (ranged from 1 nM to 10 μM) did not influence basal activity of soluble human platelet guanylate cyclase (sGC), but caused a bell-shaped inhibition of the NO-activated enzyme. Inhibition of the NO-dependent activation by isatin did not depend on a chemical nature of the NO donors. The inhibitory effects of ODC (a heme-dependent inhibitor of sGC) and isatin were non-additive suggesting that the inhibitory effect of isatin may involve the heme binding domain (possibly heme iron) and experiments with hemin revealed some isatin-dependent changes in its spectrum. Isatin also inhibited sGC activation by the allosteric activator YC-1. It is suggested that the bell shaped inhibition of the NO-dependent activation of sGC by isatin may be attributed to complex interaction of isatin with the heme binding domain and the allosteric YC-1-binding site of sGC.     

The receptor-like properties of nitric oxide-activated soluble guanylyl cyclase in intact cells

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO), and so mediates a wide range of effects (e.g. vasodilatation, platelet disaggregation and neural signalling) through the accumulation of cGMP and the engagement of various downstream targets, such as protein kinases and ion channels. Until recently, our understanding of sGC functioning has been derived exclusively from studies of the enzyme in tissue homogenates or in its purified form. Here, NO binds to the haem prosthetic group of sGC, triggering a conformational change and a large increase in catalytic activity. The potency (EC₅₀) of NO appears to be about 100–200 nM. The rate of activation of sGC by NO is rapid (milliseconds) and, in the presence of excess substrate, cGMP is formed at a constant rate; on removal of NO, sGC deactivates slowly (seconds–minutes). Recent investigation of the way that sGC behaves in its natural environment, within cells, has revealed several key differences. For example, the enzyme exhibits a rapidly desensitizing profile of activity; the potency of NO is 45 nM for the minimally-desensitized enzyme but becomes higher with time; deactivation of sGC on removal of NO is 25-fold faster than the fastest estimate for purified sGC. Overall, within cells, sGC behaves in a way that is analogous to the way that classical neurotransmitter receptors operate. The properties of cellular sGC have important implications for the understanding of NO-cGMP signalling. For example, the dynamics of the enzyme means that fluctuations in the rate of NO formation, even on subsecond time scale, will result in closely synchronized sGC activity in neighbouring cells; desensitization of sGC provides an economical way of generating a cellular cGMP signal and, in concert with phosphodiesterases, provides the basis for cGMP signal diversity, allowing different targets (outputs) to be selected from a common input (NO). Thus, despite exhibiting only limited molecular heterogeneity, cellular sGC functions in a way that introduces speed, complexity, and versatility into NO-cGMP signalling pathways.     

NO activation of guanylyl cyclase

Nitric oxide (NO)-sensitive guanylyl-cyclase (GC) is the most important receptor for the signaling molecule NO. Activation of the enzyme is brought about by binding of NO to the prosthetic heme group. By monitoring NO-binding and catalytic activity simultaneously, we show that NO activates GC only if the reaction products of the enzyme are present. NO-binding in the absence of the products did not activate the enzyme, but yielded a nonactivated species with the spectral characteristics of the active form. Conversion of the nonactivated into the activated conformation of the enzyme required the simultaneous presence of NO and the reaction products. Furthermore, the products magnesium/cGMP/pyrophosphate promoted the release of the histidine-iron bond during NO-binding, indicating reciprocal communication of the catalytic and ligand-binding domains. Based on these observations, we present a model that proposes two NO-bound states of the enzyme: an active state formed in the presence of the products and a nonactivated state. The model not only covers the data reported here but also consolidates results from previous studies on NO-binding and dissociation/deactivation of GC.     

Assay of soluble guanylate cyclase activity by isocratic high-performance liquid chromatography

A HPLC method alternative to labelled or unlabelled procedures was developed for the assay of guanylate cyclase (GC) activity. The substrate (GTP) and the product (cGMP) of the enzymatic reaction were separated in the isocratic mode on a μBondapak C₁₈ column. The activity of GC was linearly dependent on the amount of cGMP produced in the presence of sodium nitroprusside. This approach was applied to follow the purification of GC from bovine lung and to evaluate its stability in different storage conditions.     

High yield purification of soluble guanylate cyclase from bovine lung

Soluble guanylate cyclase (sGC), the main target of nitric oxide (NO), is a cytosolic, heme-containing, heterodimeric enzyme that catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3,5′-cyclic guanosine monophosphate (cGMP) and pyrophosphate (PPi) in the presence of Mg²⁺. Cyclic GMP is then involved in transmitting the NO activating signals to a variety of downstream effectors such as cyclic-nucleotide-gated channels, protein kinases, and phosphodiesterases. In this work, sGC has been purified from bovine lung. The lungs were subjected to grinding and extraction with buffer at physiological pH followed by centrifugation. The resulting solution was subjected to successive column chromatography on DEAE- and Q-Sepharose, Ceramic Hydroxyapatite, Resource Q, and GTP–agarose. The purified enzyme migrated as a two-band protein on SDS–PAGE corresponding to sGC subunits α (M_r = 77,532) and β (M_r = 70,500) and had an A_280nm/A_430nm of 1 indicating one heme per heterodimer. The yield of enzyme was 8–10 mg from 4 to 5 kg bovine lungs. V_max and K_m of non-stimulated sGC were 22 nmol/mg/min and 180 μM, respectively. Upon stimulation with the NO donor 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene, the V_max increased to 1330 nmol/mg/min while the K_m dropped to 43 μM. The quality and quantity of enzyme make it suitable for studies to probe the structure and catalytic mechanism of this enzyme and for research related to drug discovery.     

Development of a spectrophotometric assay for cyclase activity

We describe the development of a rapid colorimetric assay for soluble guanylate cyclase (sGC) activity adapted for a 96-well microplate. The assay greatly decreases the analysis time and cost over traditional methodologies based on radio- and immunoassays and high-performance liquid chromatography (HPLC) separations. The method does not demonstrate any significant interference with chemicals commonly used for sGC purification and reaction kinetics. The assay converts the inorganic pyrophosphate produced in the cyclase reaction to inorganic phosphate, which is then measured using a modified Fiske-Subbarow assay. We used the assay to compare the reaction kinetics of preparations of sGC from a commercial source with those from our lab with Mg(2+)-guanosine 5'-triphosphate (GTP) or Mn(2+)-GTP as a substrate. The commercial preparation was found to have a specific activity of around 1.5 micromol/min/mg, which is significantly lower than expected, as was the fold-activation upon addition of nitric oxide (NO). Our laboratory preparation had a higher specific activity that was consistent with results from HPLC assays. We determined that the human isoform of sGC is more active in the basal and NO forms with Mn(2)-GTP as a substrate than Mg(2+)-GTP, a feature more similar to rat lung sGC than the more commonly studied bovine lung.     

Regulation of guanylate cyclase activity by atrial natriuretic factor and protein kinase C

Summary The putative second messenger of certain atrial natriuretic factor (ANF) signal transductions is cyclic GMP. Recently, we purified a 180-kDa protein, apparently containing both ANF receptor and guanylate cyclase activities, and hypothesized that this is one of the cyclic GMP transmembrane signal transducers. The enzyme is ubiquitous and appears to be conserved. Utilizing the 180-kDa membrane guanylate cyclase, we now show that the 180-kDa guanylate cyclase is regulated in opposing fashions by two receptor signals—ANF stimulating it and protein kinase C inhibiting it. Furthermore, protein kinase C phosphorylates the 180-kDa enzyme. This suggests a novel switch on and switch off mechanism of the cyclic GMP signal transduction. Switch off represents the phosphorylation while switch on the dephosphorylation of the enzyme.     

Effects of hydrogen peroxide on relaxation through the NO/sGC/cGMP pathway in isolated rat iliac arteries

Abstract

The production of reactive oxygen species, including hydrogen peroxide (H₂O₂), is increased in diseased blood vessels. Although H₂O₂ leads to impairment of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP signaling pathway, it is not clear whether this reactive molecule affects the redox state of sGC, a key determinant of NO bioavailability. To clarify this issue, mechanical responses of endothelium-denuded rat external iliac arteries to BAY 41-2272 (sGC stimulator), BAY 60-2770 (sGC activator), nitroglycerin (NO donor), acidified NaNO₂ (exogenous NO) and 8-Br-cGMP (cGMP analog) were studied under exposure to H₂O₂. The relaxant response to BAY 41-2272 (pD₂: 6.79?±?0.10 and 6.62?±?0.17), BAY 60-2770 (pD₂: 9.57?±?0.06 and 9.34?±?0.15) or 8-Br-cGMP (pD₂: 5.19?±?0.06 and 5.24?±?0.08) was not apparently affected by exposure to H₂O₂. In addition, vascular cGMP production stimulated with BAY 41-2272 or BAY 60-2770 in the presence of H₂O₂ was identical to that in its absence. On the other hand, nitroglycerin-induced relaxation was markedly attenuated by exposing the arteries to H₂O₂ (pD₂: 8.73?±?0.05 and 8.30?±?0.05), which was normalized in the presence of catalase (pD₂: 8.59?±?0.05). Likewise, H₂O₂ exposure impaired the relaxant response to acidified NaNO₂ (pD₂: 6.52?±?0.17 and 6.09?±?0.16). These findings suggest that H₂O₂ interferes with the NO-mediated action, but the sGC redox equilibrium and the downstream target(s) of cGMP are unlikely to be affected in the vasculature.