The synthetic peptide octarphin activates soluble guanylate cyclase in macrophages

The effect of the peptide octarphin (TPLVTLFK), a selective agonist of the nonopioid β-endorphin receptor, on the activity of soluble (sGC) and membrane-bound guanylate cyclase (mGC) of peritoneal macrophages from mouse, rat, and guinea pig has been studied. It has been found that, in the concentration range of 10–1000 nM, octarphin increases the activity of sGC and has no effect on the activity of mGC in macrophages of these species. The activating effect of the peptide toward sGC was dose-dependent and was maximal at a concentration of 100 nM. These results indicate that the increase in the concentration of guanosine 3',5'-cyclophosphate in macrophages in the presence of octarphin occurs through the activation of sGC. It can be stated that the activating effect of octarphin on peritoneal macrophages is accomplished in the following way: an increase in the activity of inducible NO synthase → an increase in NO production → activation of sGC → an increase in the intracellular cGMP level.     

Relative sensitivity of soluble guanylate cyclase and mitochondrial respiration to endogenous nitric oxide at physiological oxygen concentration

Nitric oxide (NO) is a widespread biological messenger that has many physiological and pathophysiological roles. Most of the physiological actions of NO are mediated through the activation of sGC (soluble guanylate cyclase) and the subsequent production of cGMP. NO also binds to the binuclear centre of COX (cytochrome c oxidase) and inhibits mitochondrial respiration in competition with oxygen and in a reversible manner. Although sGC is more sensitive to endogenous NO than COX at atmospheric oxygen tension, the more relevant question is which enzyme is more sensitive at physiological oxygen concentration. Using a system in which NO is generated inside the cells in a finely controlled manner, we determined cGMP accumulation by immunoassay and mitochondrial oxygen consumption by high-resolution respirometry at 30 microM oxygen. In the present paper, we report that the NO EC50 of sGC was approx. 2.9 nM, whereas that required to achieve IC50 of respiration was 141 nM (the basal oxygen consumption in the absence of NO was 14+/-0.8 pmol of O2/s per 10(6) cells). In accordance with this, the NO-cGMP signalling transduction pathway was activated at lower NO concentrations than the AMPKs (AMP-activated protein kinase) pathway. We conclude that sGC is approx. 50-fold more sensitive than cellular respiration to endogenous NO under our experimental conditions. The implications of these results for cell physiology are discussed.     

Efficient expression of human soluble guanylate cyclase in Escherichia coli and its signaling-related interaction with nitric oxide

Soluble guanylate cyclase (sGC), as a nitric oxide (NO) sensor, is a critical heme-containing enzyme in NO-signaling pathway of eukaryotes. Human sGC is a heterodimeric hemoprotein, composed of a α-subunit (690 AA) and a heme-binding β-subunit (619 AA). Upon NO binding, sGC catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP). cGMP is a second messenger and initiates the nitric oxide signaling, triggering vasodilatation, smooth muscle relaxation, platelet aggregation, and neuronal transmission etc. The breakthrough of the bottle neck problem for sGC-mediated NO singling was made in this study. The recombinant human sGC β1 subunit (HsGCβ619) and its truncated N-terminal fragments (HsGCβ195 and HsGCβ384) were efficiently expressed in Escherichia coli and purified successfully in quantities. The three proteins in different forms (ferric, ferrous, NO-bound, CO-bound) were characterized by UV–vis and EPR spectroscopy. The homology structure model of the human sGC heme domain was constructed, and the mechanism for NO binding to sGC was proposed. The EPR spectra showed a characteristic of five-coordinated heme-nitrosyl species with triplet hyperfine splitting of NO. The interaction between NO and sGC was investigated and the schematic mechanism was proposed. This study provides new insights into the structure and NO-binding of human sGC. Furthermore, the efficient expression system of E. coli will be beneficial to the further studies on structure and activation mechanism of human sGC.     

Nitric oxide signaling: no longer simply on or off

Nitric oxide (NO) triggers various physiological responses in numerous tissues by binding and activating soluble guanylate cyclase (sGC) to produce the second messenger cGMP. In vivo, basal NO/cGMP signaling maintains a resting state in target cells (for example, resting tone in smooth muscle), but an acute burst of NO/cGMP signaling triggers rapid responses (such as smooth muscle relaxation). Recent studies have shown that the sGC heterodimer comprises at least four modular domains per subunit. The N-terminal heme domain is a member of the H-NOX family of domains that bind O(2) and/or NO and are conserved in prokaryotes and higher eukaryotes. Studies of these domains have uncovered the molecular basis for ligand discrimination by sGC. Other work has identified two temporally distinct states of sGC activation by NO: formation of a stable NO-heme complex results in a low-activity species, and additional NO produces a transient fully active enzyme. Nucleotides also allosterically modulate the duration and intensity of enzyme activity. Together, these studies suggest a biochemical basis for the two distinct types of NO/cGMP signal observed in vivo.     

Guanylate cyclase and the .NO/cGMP signaling pathway.

Signal transduction with the diatomic radical nitric oxide (NO) is involved in a number of important physiological processes, including smooth muscle relaxation and neurotransmission. Soluble guanylate cyclase (sGC), a heterodimeric enzyme that converts guanosine triphosphate to cyclic guanosine monophosphate, is a critical component of this signaling pathway. sGC is a hemoprotein; it is through the specific interaction of NO with the sGC heme that sGC is activated. Over the last decade, much has been learned about the unique heme environment of sGC and its interaction with ligands like NO and carbon monoxide. This review will focus on the role of sGC in signaling, its relationship to the other nucleotide cyclases, and on what is known about sGC genetics, heme environment and catalysis. The latest understanding in regard to sGC will be incorporated to build a model of sGC structure, activation, catalytic mechanism and deactivation.     

Chronic Activation of Heme Free Guanylate Cyclase Leads to Renal Protection in Dahl Salt-Sensitive Rats

The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophasphate (cGMP)-signalling pathway is impaired under oxidative stress conditions due to oxidation and subsequent loss of the prosthetic sGC heme group as observed in particular in chronic renal failure. Thus, the pool of heme free sGC is increased under pathological conditions. sGC activators such as cinaciguat selectively activate the heme free form of sGC and target the disease associated enzyme. In this study, a therapeutic effect of long-term activation of heme free sGC by the sGC activator cinaciguat was investigated in an experimental model of salt-sensitive hypertension, a condition that is associated with increased oxidative stress, heme loss from sGC and development of chronic renal failure. For that purpose Dahl/ss rats, which develop severe hypertension upon high salt intake, were fed a high salt diet (8% NaCl) containing either placebo or cinaciguat for 21 weeks. Cinaciguat markedly improved survival and ameliorated the salt-induced increase in blood pressure upon treatment with cinaciguat compared to placebo. Renal function was significantly improved in the cinaciguat group compared to the placebo group as indicated by a significantly improved glomerular filtration rate and reduced urinary protein excretion. This was due to anti-fibrotic and anti-inflammatory effects of the cinaciguat treatment. Taken together, this is the first study showing that long-term activation of heme free sGC leads to renal protection in an experimental model of hypertension and chronic kidney disease. These results underline the promising potential of cinaciguat to treat renal diseases by targeting the disease associated heme free form of sGC.     

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.     

Effects of nitroglycerin on soluble guanylate cyclase: implications for nitrate tolerance

Soluble guanylate cyclase (sGC) is a heterodimeric hemoprotein that catalyzes the conversion of GTP to cGMP. Upon binding NO to its heme cofactor, purified sGC was activated 300-fold. sGC was only activated 67-fold by nitroglycerin (GTN) and Cys; and in the absence of Cys, GTN did not activate sGC. Electronic absorption spectroscopy studies showed that upon NO binding, the Soret of ferrous sGC shifted from 431 to 399 nm. The data also revealed that activation of sGC by GTN/Cys was not via the expected ferrous heme-NO species as indicated by the absence of the 399 nm heme Soret. Furthermore, EPR studies of the reaction of GTN/Cys with sGC confirmed that no ferrous heme-NO species was formed but that there was heme oxidation. Potassium ferricyanide is known to oxidize ferrous sGC to the ferric oxidation state. Spectroscopic and activity data for the reactions of sGC with GTN alone or with K(3)Fe(CN)(6) were indistinguishable. These data suggest the following: 1) GTN/Cys do not activate sGC via GTN biotransformation to NO in vitro, and 2) in the absence of added thiol, GTN oxidizes sGC.     

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.     

Modulation of neuronal nitric oxide release by soluble guanylyl cyclase in guinea pig colon

Peripheral autonomic neurones release nitric oxide (NO) upon nerve activation. However, the regulation of neuronal NO formation is poorly understood. We used the cyclic guanosine 3',5'-monophosphate (cGMP) analogue 8-Br-cGMP, the soluble guanylyl cyclase (sGC) stimulator YC-1, the phosphodiesterase inhibitor zaprinast and the sGC inhibitor ODQ to study whether the sGC/cGMP pathway is involved in regulation of neuronal NO release in nerve plexus-containing smooth muscle preparations from guinea pig colon. Electrical stimulation of the preparation evoked release of NO/NO(-)(2). In the presence of 8-Br-cGMP, YC-1 and zaprinast (all at 10(-4) M) the NO/NO(-)(2)-release increased to 152 +/- 16% (P < 0.05), 164 +/- 37% (P < 0.05) and 290 +/- 67% (P < 0.05) of controls, respectively. Conversely, ODQ (10(-5) M) decreased the evoked release of NO/NO(-)(2) to 49 +/- 7% (P < 0.05) of controls. Our data suggest that the sGC/cGMP pathway modulates NO release. Thus it is likely that NO exerts a positive feedback on its own release from peripheral autonomic neurones.     

Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells

Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.     

Aromatic l‐amino acid decarboxylase phosphorylation and activation by PKGIαin vitro

Soluble guanylyl cyclase (sGC) is the major physiological receptor for nitric oxide (NO) throughout the central nervous system. Three different subunits form the α₁/β₁ and α₂/β₁ heterodimeric enzymes that catalyze the reaction of GTP to the second messenger cGMP. Both forms contain a prosthetic heme group which binds NO and mediates activation by NO. A number of studies have shown that NO/cGMP signaling plays a major role in neuronal cell differentiation during development of the central nervous system. In the present work, we studied regulation and expression of sGC in brain of rats during postnatal development using biochemical methods. We consistently observed a surprising decrease in cerebral NO sensitive enzyme activity in adult animals in spite of stable expression of sGC subunits. Total hemoprotein heme content was decreased in cerebrum of adult animals, likely because of an increase in heme oxygenase activity. But the loss of sGC activity was not simply because of heme loss in intact heterodimeric enzymes. This was shown by enzyme activity determinations with cinaciguat which can be used to test heme occupancy in intact heterodimers. A reduction in heterodimerization in cerebrum of adult animals was demonstrated by co‐precipitation analysis of sGC subunits. This explained the observed decrease in NO sensitive guanylyl cyclase activity in cerebrum of adult animals. We conclude that differing efficiencies in heterodimer formation may be an important reason for the lack of correlation between sGC protein expression and sGC activity that has been described previously. We suggest that heterodimerization of sGC is a regulated process that changes during cerebral postnatal development because of still unknown signaling mechanisms.     

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.     

Cyclic GMP and Nitric Oxide Synthase in Aging and Alzheimer's Disease

Cyclic guanosine monophosphate (cGMP) is an important secondary messenger synthesized by the guanylyl cyclases which are found in the soluble (sGC) and particular isoforms. In the central nervous system, the nitric oxide (NO)-sensitive sGC isoform is the major enzyme responsible for cGMP synthesis. Phosphodiesterases (PDEs) are enzymes for hydrolysis of cGMP in the brain, and they are mainly isoforms 2, 5, and 9. The NO/cGMP signaling pathway has been shown to play an important role in the process underlying learning and memory. Aging is associated with an increase in PDE expression and activity and a decrease in cGMP concentration. In addition, aging is also associated with an enhancement of neuronal NO synthase, a lowering of endothelial, and no alteration in inducible activity. The observed changes in NMDA receptor density along with the Ca²⁺/NO/cGMP pathway underscore the lower synaptic plasticity and cognitive performance during aging. This notion is in agreement with last data indicating that inhibitors of PDE2 and PDE9 improve learning and memory in older rats. In this review, we focus on recent studies supporting the role of Ca²⁺/NO/cGMP pathway in aging and Alzheimer's disease.     

Oxygen Mediates Vascular Smooth Muscle Relaxation in Hypoxia

The activation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and other ligands has been extensively investigated for many years. In the present study we considered the effect of molecular oxygen (O₂) on sGC both as a direct ligand and its affect on other ligands by measuring cyclic guanosine monophosphate (cGMP) production, as an index of activity, as well as investigating smooth muscle relaxation under hypoxic conditions. Our isolated enzyme studies confirm the function of sGC is impaired under hypoxic conditions and produces cGMP in the presence of O₂, importantly in the absence of NO. We also show that while O₂ could partially affect the magnitude of sGC stimulation by NO when the latter was present in excess, activation by the NO independent, haem-dependent sGC stimulator 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1) was unaffected. Our in vitro investigation of smooth muscle relaxation confirmed that O₂ alone in the form of a buffer bolus (equilibrated at 95% O₂/5% CO₂) had the ability to dilate vessels under hypoxic conditions and that this was dependent upon sGC and independent of eNOS. Our studies confirm that O₂ can be a direct and important mediator of vasodilation through an increase in cGMP production. In the wider context, these observations are key to understanding the relative roles of O₂ versus NO-induced sGC activation.     

Preparation of heme-free soluble guanylate cyclase

Soluble guanylate cyclase (sGC), a heterodimer consisting of alpha- and beta-subunit, is the key enzyme of the NO/cGMP signaling pathway. The heme moiety ligated to the beta-subunit via His(105) is crucial for the activation of the enzyme by NO. In addition to this NO binding capability, the heme status of the enzyme influences the activity of non-NO sGC activators and sGC inhibitors. Different sGC activity profiles were observed in the presence, absence, or the oxidized form of heme. Modulating the heme status is therefore crucial for the investigation of the mechanism of sGC activation. Here, we present a simple and reliable procedure for the removal of the heme moiety of sGC that is capable of eliminating any traces of unbound heme and detergent from the sample mixture in one single step. Samples containing 15 microg sGC and the non-ionic detergent Tween 20 (2%) were incubated at 37 degrees C for 10 min and loaded onto centrifugal ion exchange columns. After centrifugation, heme was bound entirely to the ion exchanger and could not be eluted, even after incubation with 1M NaCl. Tween 20 was found completely within the flowthrough. Heme-free sGC was eluted from the ion exchanger after application of 300 mM NaCl. The absence of the heme moiety was confirmed by UV/Vis spectra and determination of the enzymatic activity. In summary, the described procedure is suitable for the preparation of very small amounts of highly purified heme-free sGC for the investigation of the mechanism of action of different types of sGC activators.     

A cell-based nitric oxide reporter assay useful for the identification and characterization of modulators of the nitric oxide/guanosine 3',5'-cyclic monophosphate pathway

Nitric oxide (NO) plays an important role in protection against the onset and progression of various cardiovascular disorders. Therefore, the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based, and highly sensitive reporter assay for NO generated by endothelial nitric oxide synthase (eNOS). In a coculture system, NO production is indirectly monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx mediated by the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor. Using this NO reporter assay, we performed a fully automated high-throughput screening campaign for stimulators of NO synthesis. The coculture system reflects most aspects of the natural NO/cGMP pathway, namely, Ca(2+)-dependent and Ca(2+)-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The NO reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP signaling pathway.     

Identification of residues crucially involved in the binding of the heme moiety of soluble guanylate cyclase

Soluble guanylate cyclase (sGC), a heterodimeric hemeprotein, is the only receptor for the biological messenger nitric oxide (NO) identified to date and is intimately involved in various signal transduction pathways. By using the recently discovered NO- and heme-independent sGC activator BAY 58-2667 and a novel cGMP reporter cell, we could distinguish between heme-containing and heme-free sGC in an intact cellular system. Using these novel tools, we identified the invariant amino acids tyrosine 135 and arginine 139 of the beta(1)-subunit as crucially important for both the binding of the heme moiety and the activation of sGC by BAY 58-2667. The heme is displaced by BAY 58-2667 due to a competition between the carboxylic groups of this compound and the heme propionic acids for the identified residues tyrosine 135 and arginine 139. This displacement results in the release of the axial heme ligand histidine 105 and to the observed activation of sGC. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by this compound and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity.     

A real-time fluorescent assay of the purified nitric oxide receptor, guanylyl cyclase

Nitric oxide (NO) mediates intercellular signaling through activation of its receptor, soluble guanylyl cyclase (sGC), leading to elevation of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP) levels. Through this signal transduction pathway, NO regulates a diverse range of physiological effects, from vasodilatation and platelet disaggregation to synaptic plasticity. Measurement of sGC activity has traditionally been carried out using end-point assays of cGMP accumulation or by transfection of cells with “detector” proteins such as fluorescent proteins coupled to cGMP binding domains or cyclic nucleotide gated channels. Here we report a simpler approach: the use of a fluorescently labeled substrate analog, mant-GTP (2′-O-(N-methylanthraniloyl) guanosine 5′-triphosphate), which gives an increase in emission intensity after enzymatic cyclization to mant-cGMP. Activation of purified recombinant sGC by NO led to a rapid rise in fluorescence intensity within seconds, reaching a maximal 1.6- to 1.8-fold increase above basal levels. The sGC inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), eliminated the fluorescence increase due to NO, and the synergistic activator of sGC, BAY 41-2272 (3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine), increased the rate at which the maximal fluorescence increase was attained. High-performance liquid chromatography (HPLC) confirmed the formation of mant-cGMP product. This real-time assay allows the progress of purified sGC activation to be quantified precisely and, with refinement, could be optimized for use in a cellular environment.     

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.