Hemoglobin mediated nitrite activation of soluble guanylyl cyclase

Nitrite has long been known to be vasoactive when present at large concentrations but it was thought to be inactive under physiological conditions. Surprisingly, we have recently shown that supraphysiological and near physiological concentrations of nitrite cause vasodilation in the human circulation. These effects appeared to result from reduction of nitrite by deoxygenated hemoglobin. Thus, nitrite was proposed to play a role in hypoxic vasodilation. We now discuss these results in the context of nitrite reacting with hemoglobin and effecting vasodilation and present new data modeling the nitric oxide (NO) export from the red blood cell and measurements of soluble guanylate cyclase (sGC) activation. We conclude that NO generated within the interior of the red blood cell is not likely to be effectively exported directly as nitric oxide. Thus, an intermediate species must be formed by the nitrite/deoxyhemoglobin reaction that escapes the red cell and effects vasodilation.     

Purification of soluble guanylate cyclase enzyme from human platelets

Soluble guanylate cyclase enzyme was purified from human platelets. The soluble fraction of the lysed platelets was sequentially chromatographed over DEAE-sepharose, GTP-agarose and HPLC size-exclusion columns. About 0.1 mg of purified enzyme could be obtained from 2000 ml of platelet rich plasma. The purified enzyme had the specific activity of 205 nmoles cGMP/mg/min with Mn2+ as cofactor. The enzyme eluted at the 160,000 daltons position from the size-exclusion column. Electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions revealed two subunits of 83,000 and 71,000 daltons respectively.     

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.     

Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α₁β₁ has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn²⁺ but not Mg²⁺, the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α₁β₁ and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC–tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC–time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn²⁺ is a physiological sGC cofactor.     

Oxidation and loss of heme in soluble guanylyl cyclase from Manduca sexta

Oxidation and loss of heme in soluble guanylyl/guanylate cyclase (sGC), the nitric oxide receptor, is thought to be a major contributor to cardiovascular disease and is the target of compounds BAY 58-2667 and HMR1766. Using spectroelectrochemical titration, we found a truncated sGC to be highly stable in the ferrous state (234 mV) and to bind ferrous heme tightly even in the presence of NO, despite the NO-induced release of the proximal histidine. In contrast, oxidized sGC readily loses ferric heme to myoglobin (0.47 ± 0.02 h(-1)). Peroxynitrite, the presumed cellular oxidant, readily oxidizes sGC in 5 mM glutathione.     

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.     

Immunolocalization of soluble guanylyl cyclase subunits in rat kidney

Stimulation of soluble guanylyl cyclase (SGC) by nitric oxide (NO) results in the generation of cyclic guanosine monophosphate (cGMP). We recently described expression of abundant nitric oxide synthase, the enzyme by which NO is generated froml-arginine, in macula densa cells of rat kidney at the protein and mRNA level. In the present study we looked for possible targets of NO in the kidney. By light and electron microscopy, we applied polyclonal antisera against four subunits (₁,₂, ₁₂) of SGC in immunocytochemical studies of frozen sections of rat kidney. We demonstrate the presence of ₁-subunit in glomerular podocytes and of ₂-subunit in principal cells of the collecting duct. In both cell types a cytosolic localization was evident from ultrastructural analysis. Regarding the collecting duct, NO was shown by other authors to inhibit sodium reabsorption in cultured mouse cortical collecting duct principal cells. In podocytes NO may relax the contractile system of podocyte foot processes, the tone of which has been suggested to counteract the elastic distension of the capillary wall.     

Carbonic anhydrases from Trypanosoma cruzi and Leishmania donovani chagasi are inhibited by benzoxaboroles

A series of 6-substituted ureido- and thioureido-benzoxaboroles were investigated as inhibitors of carbonic anhydrases from Trypanosoma cruzi (TcCA), and Leishmania donovani chagasi (LdcCA). Both enzymes were inhibited by benzoxaboroles in the micromolar range. Preferential inhibitory potency against the β-CA LdcCA versus the α-CA TcCA was observed with submicromolar inhibitory activities. Some derivatives displayed excellent inhibitory and selectivity profile over the ubiquitous and physiological relevant human off-target hCA II. This study provides a convincing opportunity to study benzoxaborole scaffold for the design of antiprotozoan potential drugs targeting the pathogen’s carbonic anhydrases.     

Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ～150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and β sGC chains are each composed of Heme‐Nitric Oxide Oxygen (H‐NOX), Per‐ARNT‐Sim (PAS), coiled‐coil and cyclase domains. Here, we present the crystal structure of the α₁ PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the β₁ H‐NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction.     

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.     

Spontaneous and receptor-controlled soluble guanylyl cyclase activity in anterior pituitary cells

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.     

Differential inhibition of adenylyl cyclase isoforms and soluble guanylyl cyclase by purine and pyrimidine nucleotides

Mammals express nine membranous adenylyl cyclase isoforms (ACs 1-9), a structurally related soluble guanylyl cyclase (sGC) and a soluble AC (sAC). Moreover, Bacillus anthracis and Bacillus pertussis produce the AC toxins, edema factor (EF), and adenylyl cyclase toxin (ACT), respectively. 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate is a potent competitive inhibitor of AC in S49 lymphoma cell membranes. These data prompted us to study systematically the effects of 24 nucleotides on AC in S49 and Sf9 insect cell membranes, ACs 1, 2, 5, and 6, expressed in Sf9 membranes and purified catalytic subunits of membranous ACs (C1 of AC5 and C2 of AC2), sAC, sGC, EF, and ACT in the presence of MnCl(2). N-Methylanthraniloyl (MANT)-GTP inhibited C1.C2 with a K(i) of 4.2 nm. Phe-889 and Ile-940 of C2 mediate hydrophobic interactions with the MANT group. MANT-inosine 5'-[gamma-thio]triphosphate potently inhibited C1.C2 and ACs 1, 5, and 6 but exhibited only low affinity for sGC, EF, ACT, and G-proteins. Inosine 5'-[gamma-thio]triphosphate and uridine 5'-[gamma-thio]triphosphate were mixed G-protein activators and AC inhibitors. AC5 was up to 15-fold more sensitive to inhibitors than AC2. EF and ACT exhibited unique inhibitor profiles. At sAC, 2',5'-dideoxyadenosine 3'-triphosphate was the most potent compound (IC(50), 690 nm). Several MANT-adenine and MANT-guanine nucleotides inhibited sGC with K(i) values in the 200-400 nm range. UTP and ATP exhibited similar affinities for sGC as GTP and were mixed sGC substrates and inhibitors. The exchange of MnCl(2) against MgCl(2) reduced inhibitor potencies at ACs and sGC 1.5-250-fold, depending on the nucleotide and cyclase studied. The omission of the NTP-regenerating system from cyclase reactions strongly reduced the potencies of MANT-ADP, indicative for phosphorylation to MANT-ATP by pyruvate kinase. Collectively, AC isoforms and sGC are differentially inhibited by purine and pyrimidine nucleotides.     

Molecular cloning of the cDNAs coding for the two subunits of soluble guanylyl cyclase from human brain.

Complementary DNA clones corresponding to the 70 and 82 kDa subunits of soluble guanylyl cyclase from human adult brain have been isolated and sequenced. Their respective open reading frames correspond to 619 amino acids (M(r) 70,469) and 717 amino acids (M(r) 81,324). Southern blots of human genomic DNA using these clones as probes give patterns which might be compatible with the presence of more than one copy per gene, or pseudogenes, for each subunit in the human genome. Comparison of the protein sequence of the large subunit from adult brain with the subunit cloned from human fetal brain (Harteneck, C., Wedel, B., Koesling, D., Malekewitz, J., B?hme, E., and Schultz, G. (1991) FEBS Lett. 292, 217-222) revealed only 34% homology. This result demonstrates the existence of a novel large subunit isoform for soluble guanylyl cyclase in human tissues.     

Renal carbonic anhydrases are involved in the reabsorption of endogenous nitrite

Nitrite (ONO(-)) exerts nitric oxide (NO)-related biological actions and its concentration in the circulation may be of particular importance. Nitrite is excreted in the urine. Hence, the kidney may play an important role in nitrite/NO homeostasis in the vasculature. We investigated a possible involvement of renal carbonic anhydrases (CAs) in endogenous nitrite reabsorption in the proximal tubule. The potent CA inhibitor acetazolamide was administered orally to six healthy volunteers (5 mg/kg) and nitrite was measured in spot urine samples before and after administration. Acetazolamide increased abruptly nitrite excretion in the urine, strongly suggesting that renal CAs are involved in nitrite reabsorption in healthy humans. Additional in vitro experiments support our hypothesis that nitrite reacts with CO(2), analogous to the reaction of peroxynitrite (ONOO(-)) with CO(2), to form acid-labile nitrito carbonate [ONOC(O)O(-)]. We assume that this reaction is catalyzed by CAs and that nitrito carbonate represents the nitrite form that is actively transported into the kidney. The significance of nitrite reabsorption in the kidney and the underlying mechanisms, notably a direct involvement of CAs in the reaction between nitrite and CO(2), remain to be elucidated.     

Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method

Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.     

Structural and functional characterization of the dimerization region of soluble guanylyl cyclase

Soluble guanylyl cyclase (sGC) is a ubiquitous enzyme that functions as a receptor for nitric oxide. Despite the obligate heterodimeric nature of sGC, the sequence segments mediating subunit association have remained elusive. Our initial screening for relevant interaction site(s) in the most common sGC isoenzyme, alpha(1) beta(1), identified two regions in each subunit, i.e. the regulatory domains and the central regions, contributing to heterodimer formation. To map the relevant segments in the beta(1) subunit precisely, we constructed multiple N- and C-terminal deletion variants and cotransfected them with full-length alpha(1) in COS cells. Immunoprecipitation revealed that a sequence segment spanning positions 204-408 mediates binding of beta(1) to alpha(1) The same region of beta(1)[204-408] was found to promote beta /beta(1) homodimerization. Fusion of [204 beta(1)-408] to enhanced green fluorescent protein conferred binding activity to the recipient protein. Coexpression of beta(1)[204-408] with alpha(1) or beta(1) targeted the sGC subunits for proteasomal degradation, suggesting that beta(1)[204-408] forms structurally deficient complexes with alpha(1) and beta(1). Analysis of deletion constructs lacking portions of the beta(1) dimerization region identified two distinct segments contributing to alpha(1) binding, i.e. an N-terminal site covering positions 204-244 and a C-terminal site at 379-408. Both sites are crucial for sGC function because deletion of either site rendered sGC dimerization-deficient and thus functionally inactive. We conclude that the dimerization region of beta(1) extends over 205 residues of its regulatory and central domains and that two discontinuous sites of 41 and 30 residues, respectively, facilitate binding of beta(1) to the alpha(1) subunit 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.     

Dependence of soluble guanylyl cyclase activity on calcium signaling in pituitary cells

The role of nitric oxide (NO) in the stimulation of soluble guanylyl cyclase (sGC) is well established, but the mechanism by which the enzyme is inactivated during the prolonged NO stimulation has not been characterized. In this paper we studied the interactions between NO and intracellular Ca(2+) in the control of sGC in rat anterior pituitary cells. Experiments were done in cultured cells, which expressed neuronal and endothelial NO synthases, and in cells with elevated NO levels induced by the expression of inducible NO synthase and by the addition of several NO donors. Basal sGC-dependent cGMP production was stimulated by the increase in NO levels in a time-dependent manner. In contrast, depolarization of cells by high K(+) and Bay K 8644, an L-type Ca(2+) channel agonist, inhibited sGC activity. Depolarization-induced down-regulation of sGC activity was also observed in cells with inhibited cGMP-dependent phosphodiesterases but not in cells bathed in Ca(2+)-deficient medium. This inhibition was independent from the pattern of Ca(2+) signaling (oscillatory versus nonoscillatory) and NO levels, and was determined by averaged concentration of intracellular Ca(2+). These results indicate that inactivation of sGC by intracellular Ca(2+) serves as a negative feedback to break the stimulatory action of NO on enzyme activity in intact pituitary cells.     

Mapping of heme-binding domains in soluble guanylyl cyclase beta1 subunit.

Soluble guanylyl cyclase (sGC) is activated upon the interaction of NO with heme in the sGC beta1 subunit. To identify the domains contributing to heme-binding, we constructed a series of deletion mutants of the beta1 subunit, and evaluated their heme-binding capability. Deletion mutants consisting of residues 1-120 [beta1(1-120)] and 80-385 [beta1(80-385)] were the shortest mutants exhibiting heme binding among the C-terminal and N-terminal-truncated mutants, respectively. The region common to both beta1(1-120) and beta1(80-385), i.e., residues 80-120, is therefore essential for heme binding, although the residues 341-385 play an auxiliary role in heme binding. Two deletion mutants, beta1(80-195) and beta1(60-195), which include only the essential region, exhibited strong heme binding and spectral properties similar to those of the nitrosyl complex of native sGC. Thus, these heme-binding core proteins may serve as model proteins for future studies on the tertiary structure of the nitrosyl complex of sGC.