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The soluble form of guanylyl cyclase (sGC) is the main receptor for the signaling agent nitric oxide (NO), which regulates cardiomyocyte contractile function and attenuates cardiomyocyte hypertrophy. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), a regulator of vascular tone, and cardiac NO-sGC-cGMP signaling modulates cardiac stress responses, including ischemia and reperfusion (IR) injury. Here, we investigated the role of GUCY1B3 (the β subunit of sGC) in cardiomyocyte IR injury and myocardial infarction (MI) in vitro and in vivo. GUCY1B3 was upregulated in neonatal rat ventricular myocytes in response to IR injury, and GUCY1B3 overexpression restored IR-induced cell death and apoptosis. Treatment with specific inhibitors of PKCδ, PKCε, and Akt suggested that the protective effects of GUCY1B3 were mediated by PKCε/Akt signaling. In a mouse model of coronary artery ligation-induced MI, GUCY1B3 silencing aggravated MI-induced cardiac dysfunction and increased infarct size and exacerbated cardiomyocyte apoptosis in association with the inactivation of PKCε and Akt. Our results suggest that GUCY1B3 exerts cardioprotective effects through the modulation of the PKCε/Akt activity and identify a potential mechanism involved in NO-sGC-cGMP signaling in the heart.

Introduction The nitric oxide (NO), soluble guanylate cyclase (sGC), and cyclic guanosine monophosphate (cGMP) pathway is the leading pathway in penile erection. Aim To assess erectile function in a mouse model in which sGC is deficient in heme (apo-sGC) and unresponsive to NO. Methods Mutant mice (sGCβ1ki/ki) that express an sGC enzyme that retains basal activity but fails to respond to NO because of heme deficiency (apo-sGC) were used. Isolated corpora cavernosa from sGCβ1ki/ki and wild-type mice were mounted in vitro for isometric tension recordings in response to sGC-dependent and -independent vasorelaxant agents. In addition, the erectile effects of some of these agents were tested in vivo at intracavernosal injection. Main Outcome Measures In vitro and in vivo recordings of erectile responses in sGCβ1ki/ki and wild-type mice after stimulation with sGC-dependent and -independent vasorelaxant agents. Results NO-induced responses were abolished in sGCβ1ki/ki mice in vitro and in vivo. The ability of the heme-dependent, NO-independent sGC stimulator BAY 41-2272 to relax the corpora cavernosa was markedly attenuated in sGCβ1ki/ki mice. In contrast, the relaxation response to the heme- and NO-independent sGC activator BAY 58-2667 was significantly enhanced in sGCβ1ki/ki mice. The relaxing effect of sGC-independent vasorelaxant agents was similar in wild-type and sGCβ1ki/ki mice, illustrating that the observed alterations in vasorelaxation are limited to NO-sGC-cGMP–mediated processes. Conclusion Our results suggest that sGC is the sole target of NO in erectile physiology. Furthermore, this study provides indirect evidence that, in addition to sGCα1β1, sGCα2β1 is important for erectile function. In addition, the significant relaxation observed in sGCβ1ki/ki mice with the cumulative addition of the sGC activator BAY 58-2667 indicates that sGC activators might offer value in treating erectile dysfunction.

Soluble guanylyl cyclase (sGC) plays a crucial role in cyclic nucleotide signaling that regulates numerous important physiological processes. To identify new sGC inhibitors that may prevent the formation of the active catalytic domain conformation, we carried out an in silico docking screen targeting a ‘backside pocket’ of the inactive sGC catalytic domain structure. Compounds 1 and 2 were discovered to inhibit sGC even at high/saturating nitric oxide concentrations. Both compounds also inhibit the BAY 58-2667-activated sGC as well as BAY 41-2272-stimulated sGC activity. Additional biochemical analyses showed that compound 2 also inhibits the isolated catalytic domain, thus demonstrating functional binding to this domain. Both compounds have micromolar affinity for sGC and are potential leads to develop more potent sGC inhibitors.

Purpose The nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway regulates aqueous humor outflow and therefore, intraocular pressure. We investigated the pharmacologic effects of the soluble guanylate cyclase (sGC) stimulator IWP-953 on primary human trabecular meshwork (HTM) cells and conventional outflow facility in mouse eyes. Methods Cyclic GMP levels were determined in vitro in HEK-293 cells and four HTM cell strains (HTM120/HTM123: predominantly myofibroblast-like phenotype, HTM130/HTM141: predominantly endothelial-like phenotype), and in HTM cell culture supernatants. Conventional outflow facility was measured following intracameral injection of IWP-953 or DETA-NO using a computerized pressure-controlled perfusion system in enucleated mouse eyes ex vivo. Results IWP-953 markedly stimulated cGMP production in HEK-293 cells in the presence and absence of DETA-NO (half maximal effective concentrations: 17 nM, 9.5 μM). Similarly, IWP-953 stimulated cGMP production in myofibroblast-like HTM120 and HTM123 cells, an effect that was greatly amplified by the presence of DETA-NO. In contrast, IWP-953 stimulation of cGMP production in endothelial-like HTM130 and HTM141 cells was observed, but was markedly less prominent than in HTM120 and HTM123 cells. Notably, cGMP was found in all HTM culture supernatants, following IWP-953/DETA-NO stimulation. In paired enucleated mouse eyes, IWP-953 at 10, 30, 60, and 100 μM concentration-dependently increased outflow facility. This effect (89.5%) was maximal at 100 μM (P = 0.002) and in magnitude comparable to DETA-NO at 100 μM (97.5% increase, P = 0.030). Conclusions These data indicate that IWP-953, via modulation of the sGC-cGMP pathway, increases aqueous outflow facility in mouse eyes, suggesting therapeutic potential for sGC stimulators as novel ocular hypotensive drugs.

The influence of (1H-1,2,3-triazol-1-yl)-1,2,5-oxadiazole derivatives: 4-amino-3-(5-methyl-4- ethoxycarbonyl-(1H-1,2,3-triazol-1-yl)-1,2,5-oxadiazole (TF4CH3) and 4,4'-bis(5-methel-4-ethoxycarbonyl-1H- 1,2,3-triazol-1-yl)-3,3'-azo-1,2,5-oxadiazole (2TF4CH3) on stimulation of human platelet soluble guanylate cyclase by YC-1, NO-donors (sodium nitroprusside, SNP, and spermine NONO) and on a synergistic increase of NO-dependent enzyme activation in the presence of YC-1 has been investigated. Both compounds increased guanylate cyclase activation by YC-1, potentiated guanylate cyclase stimulation by NO-donors and increased the synergistic effect of YC-1 on NO-dependent activation of soluble guanylate cyclase. The similarity in the properties of the examined TF4CH3 and 2TF4CH3 with that of YC-1 and the possible mechanism underlying the revealed properties of compounds used are discussed.Issledovano vliianie proizvodnykh 1,2,3-(triazolil)-1,2,5-oksidiazola: (4-amino-3-(5-metil-4- étoksikarbonil-1N-(1,2,3-triazol-1-il)-1,2,5-oksadiazol (TF 4 SN 3 ) i 4,4’-bis(5-metil-4- étoksikarbonil-1N-(1,2,3-triazol-1-il)-3,3’-azo-1,2,5-oksadiazol (2TF 4 SN 3 ) na stimuliatsiiu rastvorimoĭ guanilattsiklazy trombotsitov cheloveka YC-1, NO-donorami (nitroprussidom natriia i spermin nanoatom) i na sinergichnoe uvelichenie NO-zavisimoĭ aktivatsii fermenta v prisutstvii YC-1. Oba soedineniia usilivali aktivatsiiu guanilattsiklazy YC-1, potentsirovali stimuliatsiiu fermenta NO-donorami i uvelichivali sinergichnyĭ éffekt YC-1 na NO-zavisimuiu aktivatsiiu guanilattsiklazy. Obsuzhdaetsia analogiia v svoĭstvakh issledovannykh proizvodnykh 1,2,3-triazolil-1,2,5-oksadiazola s YC-1 i vozmozhnyĭ mekhanizm, lezhashchiĭ v osnove vyiavlennykh svoĭstv izuchennykh soedineniĭ.

Soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. NO binds to the heme of sGC to catalyze the synthesis of the second messenger cGMP, which plays a critical role in several physiological processes. However, the molecular mechanism for sGC to mediate the NO signaling remains unclear. Here fluorophore FlAsH-EDT2 and fluorescent proteins were employed to study the NO-induced sGC activation. FlAsH-EDT2 labeling study revealed that NO binding to the H-NOX domain of sGC increased the distance between H-NOX and PAS domain and the separation between H-NOX and coiled-coil domain. The heme pocket conformation changed from “closed” to “open” upon NO binding. In addition, the NO-induced conformational change of sGC was firstly investigated in vivo through fluorescence lifetime imaging microscopy. The results both in vitro and in vivo indicated the conformational change of the catalytic domain of sGC from “open” to “closed” upon NO binding. NO binding to the heme of H-NOX domain caused breaking of Fe-N coordination bond, initiated the domain moving and conformational change, induced the allosteric effect of sGC to trigger the NO-signaling from H-NOX via PAS & coiled-coil to the catalytic domain, and ultimately stimulates the cyclase activity of sGC.

Cyclic nucleotides such as 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

Objective: Vascular dysfunction plays a central role in sepsis, and it is characterized by hypotension and hyporesponsiveness to vasoconstrictors. Nitric oxide is regarded as a central element of sepsis vascular dysfunction. The high amounts of nitric oxide produced during sepsis are mainly derived from the inducible isoform of nitric oxide synthase 2. We have previously shown that nitric oxide synthase 2 levels decrease in later stages of sepsis, whereas levels and activity of soluble guanylate cyclase increase. Therefore, we studied the putative role of other relevant nitric oxide sources, namely, the neuronal (nitric oxide synthase 1) isoform, in sepsis and its relationship with soluble guanylate cyclase. We also studied the consequences of nitric oxide synthase 1 blockade in the hyporesponsiveness to vasoconstrictors. Design: Randomized controlled prospective experimental study. Setting: Academic research laboratory. Subjects: Female Wistar rats submitted to cecal ligation and puncture method. Interventions: 1) Six, 12, and 24 hours after cecal ligation and puncture, vascular reactivity to phenylephrine (3 and 30 nmol/kg) before and after 7-nitroindazole (45 μmol/kg, s.c.) or aminoguanidine (30 μmol/kg, s.c.) administration was evaluated. 2) Protein levels and interaction between nitric oxide synthase 1 and soluble guanylate cyclase were determined. 3) Six, 12, and 24 hours after cecal ligation and puncture, thoracic aorta segments were stimulated with phenylephrine in the presence or absence of 7-nitroindazole and cyclic guanosine monophosphate accumulation was determined. 4) After 24 hours of cecal ligation and puncture, norepinephrine was infused (10 μg/kg/min) in the presence or absence of 7-nitroindazole or S-methyl-L-thiocitrulline (1 μmol/kg, IV) and mean arterial pressure was registered. Measurements and Main Results: 1) Both nitric oxide synthase 1 and soluble guanylate cyclase are expressed in higher levels in vascular tissues during sepsis; 2) both proteins physically interact and nitric oxide synthase 1 blockade inhibits cyclic guanosine monophosphate production; 3) pharmacological blockade of nitric oxide synthase 1 using 7-nitroindazole or S-methyl-L-thiocitrulline reverts the hyporesponsiveness to phenylephrine and increases the vasoconstrictor effect of norepinephrine and phenylephrine. Conclusions: Sepsis induces increased expression and physical association of nitric oxide synthase 1/soluble guanylate cyclase and a higher production of cyclic guanosine monophosphate that together may help explain sepsis-induced vascular dysfunction. In addition, selective inhibition of nitric oxide synthase 1 restores the responsiveness to vasoconstrictors. Therefore, inhibition of nitric oxide synthase 1 (and possibly soluble guanylate cyclase) may represent a valuable alternative to restore the effectiveness of vasopressor agents during late sepsis.

The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells including soluble guanylate cyclase (sGC). hsp90 associates with the heme-free (apo) sGC-β1 subunit and helps to drive heme insertion during maturation of sGC to its NO-responsive active form. Here, we found that NO caused apo-sGC-β1 to rapidly and transiently dissociate from hsp90 and associate with sGC-α1 in cells. This NO response (i) required that hsp90 be active and that cellular heme be available and be capable of inserting into apo-sGC-β1; (ii) was associated with an increase in sGC-β1 heme content; (iii) could be mimicked by the heme-independent sGC activator BAY 60-2770; and (iv) was followed by desensitization of sGC toward NO, sGC-α1 disassociation, and reassociation with hsp90. Thus, NO promoted a rapid, transient, and hsp90-dependent heme insertion into the apo-sGC-β1 subpopulation in cells, which enabled it to combine with the sGC-α1 subunit to form the mature enzyme. The driving mechanism likely involves conformational changes near the heme site in sGC-β1 that can be mimicked by the pharmacologic sGC activator. Such dynamic interplay between hsp90, apo-sGC-β1, and sGC-α1 in response to NO is unprecedented and represent new steps by which cells can modulate the heme content and activity of sGC for signaling cascades.

The complete structure of the assembled domains of nitric oxide-sensitive guanylate cyclase (NOsGC) remains to be determined. It is also unknown how binding of NO to heme in guanylate cyclase is communicated to the catalytic domain. In the current study the conformational change of guanylate cyclase on activation by NO was studied using FRET. Endogenous tryptophan residues were used as donors, the substrate analog 2'-Mant-3'-dGTP as acceptor. The enzyme contains five tryptophan residues distributed evenly over all four functional domains. This provides a unique opportunity to detect the movement of the functional domains relative to the substrate-binding catalytic region. FRET measurements indicate that NO brings tryptophan 22 in the αB helix of the β1 heme NO binding domain and tryptophan 466 in the second short helix of the α1 coiled-coil domain closer to the catalytic domain. We propose that the respective domains act as a pair of tongs forcing the catalytic domain into the nitric oxide-activated conformation.

Nitric oxide (NO), a signaling molecule in the cardiovascular system, has been receiving increasing attention since Furchgott, Ignarro, and Murad were awarded the Nobel Prize in Physiology and Medicine for the discovery in 1998. Soluble guanylate cyclase (sGC), as an NO receptor, is a key metalloprotein in mediating NO signaling transduction. sGC is activated by NO to catalyze the conversion of guanosine 5′-triphosphate (GTP) to cyclic guanylate monophosphate (cGMP). The dysfunction of NO signaling results in many pathological disorders, including several cardiovascular diseases, such as arterial hypertension, pulmonary hypertension, heart failure and so on. Significant advances in its structure, function, mechanism, and physiological and pathological roles have been made throughout the past 15 years. We herein review the progress of sGC on structural, functional investigations, as well as the proposed activation/deactivation mechanism. The heme-dependent sGC stimulators and heme-independent sGC activators have also been summarized briefly.

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.

Background & Aims It is not clear how nitric oxide (NO) released from enteric neurons relaxes gastrointestinal (GI) smooth muscle. In analogy to the vascular system, NO might directly induce relaxation of smooth muscle cells (SMCs) by acting on its receptor, NO-sensitive guanylyl cyclase (NO-GC). Alternatively, intermediate cells, such as the interstitial cells of Cajal (ICCs), might detect nitrergic signals to indirectly regulate smooth muscle tone, and thereby regulate the motor function of the GI tract. We investigated the role of ICCs and SMCs in nitrergic relaxation using mice with cell-specific disruption of the gene encoding the β 1 subunit of NO-GC ( GUCY1B3 ). Methods We created mice that lack NO-GC specifically in SMCs (SM-guanylyl cyclase knockout [GCKO]), ICCs (ICC-GCKO), or both (SM/ICC-GCKO). We investigated the effects of exogenous and endogenous NO on murine fundus using isometric force studies. Total gut transit time was measured to monitor the functional consequences of NO-GC deletion on GI motility in vivo. Results NO-GC is expressed in ICC and SMC. Deletion of the NO receptor from SMCs incompletely reduced NO-induced fundus relaxation, which was hardly affected after ICC-specific deletion. Gut transit time did not change in SM-GCKO or ICC-GCKO mice compared with control mice. However, nitrergic relaxation was not observed in SM/ICC-GCKO mice, which had increased gut transit time compared with controls. Conclusions In mice, NO-GC is the only NO receptor to relax the fundus; deletion of NO-GC from the combination of SMCs and ICCs blocks nitrergic signaling. Therefore, ICCs and SMCs jointly mediate the relaxant effect of enteric NO.

1-Nitro-2-phenylethane is the first organic NO2-containing molecule isolated from plants. It possesses interesting hypotensive, bradycardic, and vasodilator properties, but the mode by which it induces vasorelaxation is still unknown. The underlying mechanism involved in the vasodilator effect of 1-nitro-2-phenylethane was investigated in rat aorta. The vasorelaxant effects of 1-nitro-2-phenylethane did not depend on endothelial layer integrity, and the effects were refractory to L-NG-nitroarginine methyl ester (L-NAME)-induced nitric oxide synthase inhibition. Vasorelaxation was similarly resistant to treatment with indomethacin, cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride (MDL-12330A), and KT5720, indicating that neither prostaglandin release nor adenylyl cyclase activation is involved. Conversely, methylene blue- and ODQ-induced guanylate cyclase inhibition reduced the vasorelaxation induced by 1-nitro-2-phenylethane. The pharmacological blockade of K+ channels with tetraethylammonium, glybenclamide, and 4-aminopyridine also blunted vasorelaxation induced by 1-nitro-2-phenylethane. The effects of 1-nitro-2-phenylethane were reversed by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and comparable to the effects induced by sodium nitroprusside. In silico analysis using an Ns H-NOX subunit of guanylate cyclase revealed a pocket on the macromolecule surface where 1-nitro-2-phenylethane preferentially docked. In vitro, 1-nitro-2-phenylethane increased cyclic guanosine 3′,5′-monophosphate (cGMP) levels in rat aortic rings, an effect also reversed by ODQ. In conclusion, 1-nitro-2-phenylethane produces vasodilator effects by stimulating the soluble guanylate cyclase-cGMP pathway.

Nitric oxide (NO) elicits a wide variety of physiological responses by binding to the heme in soluble guanylate cyclase (sGC) to stimulate cGMP production. Although nucleotides, such as ATP or GTP analogues, have been reported to regulate the signaling of NO binding from the heme site to the catalytic site, the other regulatory functions of nucleotides remain unexamined. Among the nucleotides tested, we found that 2'-d-3'-GMP acted as a potent noncompetitive inhibitor with respect to Mn-GTP, when the ferrous enzyme combined with NO, CO, or allosteric activator BAY 41-2272. 2'-d-3'-GMP also displayed nearly identical patterns of inhibition for the ferric enzyme, in which the binding of N(3)(-) or BAY 41-2272 significantly increased the inhibitory effects of the nucleotide. Equilibrium dialysis measurements using the CO-ligated enzyme in the presence of allosteric activators demonstrated that 2'-d-3'-GMP exclusively binds to the catalytic site of sGC. Furthermore, the affinity of 2'-d-3'-GMP for the enzyme was found to increase upon addition of foscarnet, an analogue of PP(i). These findings together with other kinetic results imply that 2'-d-3'-GMP acts as a P-site inhibitor probably by forming a dead-end complex, sGC-2'-d-3'-GMP-PP(i), in the catalytic reaction. The formation of the complex of the enzyme with 2'-d-3'-GMP does not seem to be associated with changes in the Fe-proximal His bond strength, because the CO coordination state or the redox potentials of the enzyme-heme complex are virtually unaffected.

Background Direct interaction between Red blood cells (RBCs) and platelets is known for a long time. The bleeding time is prolonged in anemic patients independent of their platelet count and could be corrected by transfusion of RBCs, which indicates that RBCs play an important role in hemostasis and platelet activation. However, in the last few years, opposing mechanisms of platelet inhibition by RBCs derived nitric oxide (NO) were proposed. The aim of our study was to identify whether RBCs could produce NO and activate soluble guanylate cyclase (sGC) in platelets. Methods To test whether RBCs could activate sGC under different conditions (whole blood, under hypoxia, or even loaded with NO), we used our well-established and highly sensitive models of NO-dependent sGC activation in platelets and activation of purified sGC. The activation of sGC was monitored by detecting the phosphorylation of Vasodilator Stimulated Phosphoprotein (VASPS239) by flow cytometry and Western blot. ANOVA followed by Bonferroni’s test and Student’s t-test were used as appropriate. Results We show that in the whole blood, RBCs prevent NO-mediated inhibition of ADP and TRAP6-induced platelet activation. Likewise, coincubation of RBCs with platelets results in strong inhibition of NO-induced sGC activation. Under hypoxic conditions, incubation of RBCs with NO donor leads to Hb-NO formation which inhibits sGC activation in platelets. Similarly, RBCs inhibit activation of purified sGC, even under conditions optimal for RBC-mediated generation of NO from nitrite. Conclusions All our experiments demonstrate that RBCs act as strong NO scavengers and prevent NO-mediated inhibition of activated platelets. In all tested conditions, RBCs were not able to activate platelet or purified sGC.

Oxidative stress interferes with nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) signalling pathway through reduction of endogenous NO and formation of the strong intermediate oxidant peroxynitrite and leads to vascular dysfunction. We evaluated the effects of oral treatment with NO- and heme-independent sGC activator cinaciguat on peroxynitrite-induced vascular dysfunction in rat aorta. Sprague-Dawley rats were treated orally 2 times at an interval of 17 hours with vehicle or with cinaciguat (10 mg/kg). One hour after the last treatment, the animals were anesthetized, the thoracic aorta was removed, and the aortic segment preparations were incubated with and without the reactive oxidant peroxynitrite (200 µmol/L, 30 minutes). Endothelium-dependent (acetylcholine), -independent (sodium nitroprusside) vasorelaxations were investigated, and histopathological examination was performed. Incubation of aortic rings with peroxynitrite significantly attenuated the maximal endothelium-dependent relaxation ( Rmax) to acetylcholine (peroxynitrite, 44.5% ± 5.9% vs control, 93.2% ± 2.0%, P < .05) and decreased pD2 values (-logEC50, EC50 being the concentration of acetylcholine that elicited 50% of the maximal response) for the concentration–response curves as compared to control segments. Treatment of rats with cinaciguat significantly improved the decreased acetylcholine-induced vasorelaxation after exposure of aortic rings to peroxynitrite (cinaciguat + peroxynitrite, 67.1% ± 3.5% vs peroxynitrite, 44.5% ± 5.9%, P < .05). Incubation of aortic segments with peroxynitrite caused a significant shift of the sodium nitroprusside concentration–response curves to the right without any alterations in the Rmax. Moreover, exposure of aortic rings to peroxynitrite resulted in increased nitro-oxidative stress and DNA breakage which were improved by cinaciguat. Treatment of rats with cinaciguat significantly increased intracellular cGMP levels in the aortic wall. Our results show under conditions of nitro-oxidative stress when signalling in the NO/sGC/cGMP pathway is impaired, acute activation of sGC by cinaciguat might be advantageous in the treatment of endothelial dysfunction in cardiovascular disease.

To evaluate the involvement K+ channels in the relaxation induced by exogenous nitric oxide, after preincubation with L-arginine analogue L-NA in the human nonpregnant myometrium. The activity of myometrial strips, obtained from 60 premenopausal hysterectomised women, mounted in an organ bath was recorded under isometric conditions using force transducers with digital output. Concentration-response curves to DEA/NO after inhibition of endogenous NO were constructed in the absence and presence of soluble granulate cyclase and K+ channels’ blockers. The responses were quantified by calculating the area under the curve, the amplitude and frequency of the contractions. The inhibition of NOS results in slight but significant attenuation of the myometrium strips response to DEA/NO. Pre-treatment with both sGC inhibitors after preincubation with L-NA did not counteract the DEA-NOinduced relaxation of the spontaneous contractions of the myometrial strips. Application of blockers of different types of K+ channels to the myometrial strips significantly attenuated relaxing effect of cumulative DEA/NO administration in all cases. The present data indicate that even when endogenous production of NO is inhibited, the DEA/NO induced relaxation of human non-pregnant myometrium without involving the cGMP pathway.

In plants, nitric oxide-mediated 3′, 5′- cyclic guanosine monophosphate (cGMP) synthesis plays an important role during pathogenic stress response, stomata closure upon osmotic stress, the development of adventitious roots and transcript regulation. The nitric oxide-cGMP dependent pathway is well characterized in mammals. The binding of nitric oxide to soluble guanylate cyclase enzymes (GCs) initiates the synthesis of cGMP from guanosine triphosphate. The produced cGMP alters various cellular responses, such as the function of protein kinase activity, cyclic nucleotide gated ion channels and cGMP-regulated phosphodiesterases. The signal generated by the second messenger is terminated by 3′, 5′- cyclic nucleotide phosphodiesterase (PDEs) enzymes that hydrolyze cGMP to a non cyclic 5′- guanosine monophosphate. To date, no homologues of mammalian cGMP-synthesizing and degrading enzymes have been found in higher plants. In the last decade, six receptor proteins from Arabidopsis thaliana have been reported to have guanylate cyclase activity in vitro. Of the six receptors, one was shown to be a nitric oxide dependent guanylate cyclase enzyme (NOGC1). However, the role of these proteins in planta remains to be elucidated. Enzymes involved in the degradation of cGMP remain elusive, albeit, PDE activity has been detected in crude protein extracts from various plants. Additionally, several research groups have partially purified and characterized PDE enzymatic activity from crude protein extracts. In this review, we focus on presenting advances toward the identification of enzymes involved in the cGMP metabolism pathway in higher plants.

In this study, we reported that a bioluminescent assay for nitric oxide (NO) was developed utilizing the biological enzyme activity of soluble guanylate cyclase (sGC) and a bioluminescent assay for pyrophosphate involving the pyruvate phosphate dikinase (PPDK)-luciferin/Luciferase reaction. Pyrophosphate (PPi), which is released concomitantly when NO binds to soluble guanylate cyclase, was measured employing the PPDK/Luciferase reaction. NO binds to soluble guanylate cyclase specifically; thus, this assay is undisturbed by nitrate anhydride, or . Consequently, the measurable range of NO obtained in the proposed method is 200–20,000 nM; the detection limit was 200 fmol/assay. NO released from nitrate medicine was measured utilizing this assay. As a result, NO released from isosorbide nitrate was detected. In conclusion, we suggest in this report that the technique for NO might be suitable as a quality check method for the pharmacodynamic action of nitrate medicines and in development of new medicines.

A series of acidic triazoles with activity as soluble guanylate cyclase stimulators is described. Incorporation of the CF3 triazole improved the overall physicochemical and drug-like properties of the molecule and is exemplified by compound 25.

Vitamin C, or ascorbic acid, decreases paracellular endothelial permeability in a process that requires rearrangement of the actin cytoskeleton. To define the proximal mechanism of this effect, we tested whether it might involve enhanced generation and/or sparing of nitric oxide (NO) by the vitamin. EA.hy926 endothelial cells cultured on semi-porous filter supports showed decreased endothelial barrier permeability to radiolabeled inulin in response to exogenous NO provided by the NO donor spermine NONOATE, as well as to activation of the downstream NO pathway by 8-bromo-cyclic GMP, a cell-penetrant cyclic GMP analog. Inhibition of endothelial nitric oxide synthase (eNOS) with N(ω)-nitro-l-arginine methyl ester increased endothelial permeability, indicating a role constitutive NO generation by eNOS in maintaining the permeability barrier. Inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one also increased endothelial permeability and blocked barrier tightening by spermine NONOATE. Loading cells with what are likely physiologic concentrations of ascorbate decreased endothelial permeability. This effect was blocked by inhibition of either eNOS or guanylate cyclase, suggesting that it involved generation of NO by eNOS and subsequent NO-dependent activation of guanylate cyclase. These results show that endothelial permeability barrier function depends on constitutive generation of NO and that ascorbate-dependent tightening of this barrier involves maintaining NO through the eNOS/guanylate cyclase pathway.

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.

The Ca(2+)-modulated ONE-GC membrane guanylate cyclase is a central component of the cyclic GMP signaling in odorant transduction. It is a single transmembrane spanning modular protein. Its intracellular region contains Ca(2+) sensor recognition domains linked to GCAP1 and to neurocalcin delta, and a catalytic module. These domains sense increments in free Ca(2+) and stimulate the catalytic module. The present study makes three significant mechanistic advancements. First, to date no ligand for the extracellular (ext) domain is known, for this reason ONE-GC has been deemed as an orphan receptor. The present study identifies its ligand. Uroguanylin stimulates ONE-GC through its ext domain. Second, so far no ligand is known that directly stimulates the catalytic module of any membrane guanylate cyclase. The presented evidence shows that in the presence of the semimicromolar range of free Ca(2+), neurocalcin binds to the catalytic module and stimulates ONE-GC. Thus, ONE-GC has trimodal regulation, two occurring intracellularly and one extracellularly. Third, guanylin, a urine odorant, does not directly stimulate ONE-GC. This challenges the proposed hypothesis that the guanylin odorant signal occurs via ONE-GC [T. Leinders-Zufall, R.E. Cockerham, S. Michalakis, M. Biel, D.L. Garbers, R.R. Reed, F. Zufall, S.D. Munger, Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium, Proc. Natl. Acad. Sci. USA. 104 (2007) 14507-14512].

Nitric oxide (NO) is a physiologically relevant activator of the hemoprotein soluble guanylate cyclase (sGC). In the presence of NO, sGC is activated several hundredfold above the basal level by a mechanism that remains to be elucidated. The heme ligand n-butyl isocyanide (BIC) was used to probe the mechanism of NO activation of sGC. Electronic absorption spectroscopy was used to show that BIC binds to the sGC heme, forming a 6-coordinate complex with an absorbance maximum at 429 nm. BIC activates sGC 2-5-fold, and synergizes with the allosteric activator YC-1, to activate the enzyme 15-25-fold. YC-1 activates the sGC-BIC complex, and leads to an increase in both the Vmax and Km. BIC was also used to probe the mechanism of NO activation. The activity of the sGC-BIC complex increases 15-fold in the presence of NO, without displacing BIC at the heme, which is consistent with previous reports that proposed the involvement of a non-heme NO binding site in the activation process.

Nitric oxide (NO) is a mediator of copious biological processes, in many cases through the production of cGMP from the enzyme nitric oxide-sensitive guanylyl cyclase. Natriuretic peptides also elevate cGMP, often with distinct biological effects, raising the issue of how specificity is achieved. Here we show that a recently described alpha(2)beta(1) isoform of guanylyl cyclase is expressed in a number of epithelia, where it is localized to the apical plasma membrane. We measured the functional properties of the alpha(2)beta(1) isoform by utilizing the NO-dependent activation of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR), which occurs by phosphorylation via the membrane-bound type II isoform of cGMP-dependent protein kinase. We found that cGMP generated by NO activation of the alpha(2)beta(1) isoform of guanylyl cyclase is an exceptionally efficient mediator of nitric oxide action on membrane targets, activating CFTR far more effectively than the cytoplasmically located alpha(1)beta(1) guanylyl cyclase isoform. Targeting the alpha(1)beta(1) isoform of guanylyl cyclase to the membrane also dramatically enhanced the effects of nitric oxide on CFTR within the membrane. This was not due to increased enzymatic activity of guanylyl cyclase in a membrane location, but to production of a localised membrane pool of cGMP by membrane-localized NO-dependent guanylyl cyclase that was resistant to degradation by phosphodiesterases. Selective effects of cGMP produced from this enzyme in response to NO are directed at membrane targets and suggest that drugs selectively activating or inhibiting this alpha(2)beta(1) isoform of guanylyl cyclase may have unique pharmacological properties.

Soluble guanylate cyclase (sGC) is activated by the known benzylindazole derivative YC-1 [1-benzyl-3-(5'-hydroxymethyl-2'-furyl)-indazole]. YC-1 also acts synergistically with CO, activating sGC to a level comparable to that achieved upon binding of nitric oxide, the endogenous activator of sGC. We here describe the synthesis of a YC-1 phosphonate analogue with improved aqueous solubility as well as its effects on sGC.

The influence of polyamines (putrescine, spermidine, and spermine) on the activity of human platelet soluble guanylate cyclase and the stimulation of the enzyme by sodium nitroprusside (SNP), YC-1 and their combination was investigated. All these polyamines stimulated the guanylate cyclase activity and potentiated its activation by sodium nitroprusside. The stimulatory effects of sodium nitroprusside and putrescine (or spermine) were addidive; spermidine produced a synergistic activation and increased the additive effect. All the polyamines inhibited the enzyme activation by YC-1 and decreased the synergistic activation of SNP-stimulated guanylate cyclase activity by YC-1 with nearly the same potency. The ability of the investigated polyamines to potentiate and to increase synergistically (similar to to YC-1, but less effective) NO-dependent activation of soluble guanylate cyclase represents a new biochemical effect of these compounds; this effect should be taken into consideration, especially due to the endogenous nature of polyamines. The data obtained suggest, that specific biological functions of polyamines in the processes of growth and differentiation of cells may be also related to the ability of compounds to activate soluble guanylate cyclase and to increase intracellular cGMP level.

The objective of this study was to examine the effects of manipulating the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway on bovine oocyte nuclear maturation in vitro. Cumulus-enclosed oocytes (CEO) were recovered from abattoir-derived ovaries and cultured in M199+FCS for 7 or 21h in the presence of various molecules affecting the NO/cGMP pathway, and then fixed and stained for evaluation of the stage of nuclear maturation. Cyclic GMP levels were also measured in cumulus-oocyte complexes after 3 and 6 h of culture. The iNOS inhibitor, aminoguanidine (AG, 10 and 50 mM) and the NO donor sodium nitroprusside (SNP, 100 and 500 microM) significantly inhibited GVBD after 7h of culture. However, a lower concentration of SNP (0.01 microM) stimulated GVBD. The inhibitory effects of AG and SNP were reversible, indicating that they were not toxic effects. Although SNP (500 microM) increased cGMP levels in cumulus-oocyte complexes after 3 h of culture, the inhibitor of soluble guanylate cyclase ODQ and the protein kinase G (PKG) inhibitor KT5823 did not reverse the inhibitory effect of SNP on meiosis, suggesting that SNP does not inhibit meiosis through the cGMP/PKG pathway. Similarly, an analogue of cGMP (8-Bromo-cGMP 0.5, 1, 3, and 6 mM), as well as activation of guanylate cyclase with Protoporphyrin IX or atrial natriuretic peptide, or inhibition of the enzyme with ODQ, did not have any significant effect on GVBD after 7 h of culture, supporting the idea that the effects of AG and SNP were not due to altered cGMP levels. Atrial natriuretic peptide, Protoporphyrin IX and SNP 500 microM increased cGMP levels after 3 h but not 6 h of culture. In conclusion, soluble and particulate guanylate cyclases could be activated in bovine cumulus-oocyte complexes, but accumulation of cGMP was probably not responsible for the effects of NO on meiosis.

The heme cofactor in soluble guanylate cyclase (sGC) is a selective receptor for NO, an important signaling molecule in eukaryotes. The sGC heme domain has been localized to the N-terminal 194 amino acids of the beta1 subunit of sGC and is a member of a family of conserved hemoproteins, called the H-NOX family (Heme-Nitric Oxide and/or OXygen-binding domain). Three new members of this family have now been cloned and characterized, two proteins from Legionella pneumophila (L1 H-NOX and L2 H-NOX) and one from Nostoc punctiforme (Np H-NOX). Like sGC, L1 H-NOX forms a 5-coordinate Fe(II)-NO complex. However, both L2 H-NOX and Np H-NOX form temperature-dependent mixtures of 5- and 6-coordinate Fe(II)-NO complexes; at low temperature, they are primarily 6-coordinate, and at high temperature, the equilibrium is shifted toward a 5-coordinate geometry. This equilibrium is fully reversible with temperature in the absence of free NO. This process is analyzed in terms of a thermally labile proximal Fe(II)-His bond and suggests that in both the 5- and 6-coordinate Fe(II)-NO complexes of L2 H-NOX and Np H-NOX, NO is bound in the distal heme pocket of the H-NOX fold. NO dissociation kinetics for L1 H-NOX and L2 H-NOX have been determined and support a model in which NO dissociates from the distal side of the heme in both 5- and 6-coordinate complexes.

Vascular endothelial growth factor-A (VEGF), which binds to both VEGF receptor-1 (Flt1) and VEGFR-2 (KDR/Flk-1), requires nitric oxide (NO) to induce angiogenesis in a cGMP-dependent manner. Here we show that VEGF-E, a VEGFR-2-selective ligand stimulates NO release and tube formation in human umbilical vein endothelial cells (HUVEC). Inhibition of phospholipase Cgamma (PLCgamma) with U73122 abrogated VEGF-E induced endothelial cell migration, tube formation and NO release. Inhibition of endothelial nitric oxide synthase (eNOS) using l-NNA blocked VEGF-E-induced NO release and angiogenesis. Pre-incubation of HUVEC with the soluble guanylate cyclase inhibitor, ODQ, or the protein kinase G (PKG) inhibitor, KT-5823, had no effect on angiogenesis suggesting that the action of VEGF-E is cGMP-independent. Our data provide the first demonstration that VEGFR-2-mediated NO signaling and subsequent angiogenesis is through a mechanism that is dependent on PLCgamma but independent of cGMP and PKG.

In the study of anti-proinflammation by 7-[2-[4-(2-chlorobenzene)piperazinyl] ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-3), exposure of rat tracheal smooth muscle cells (TSMCs) to tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increased the expression of inducible nitric-oxide synthase (iNOS) and NO production and decreased the expression of soluble guanylate cyclase alpha1 (sGCalpha1), soluble guanylate cyclase beta1 (sGCbeta1), protein kinase G (PKG), and the release of cGMP in TSMCs. The cell-permeable cGMP analog 8-Br-cGMP, xanthine-based KMUP-1 and KMUP-3, and the phosphodiesterase 5 inhibitor zaprinast all inhibited TNF-alpha-induced increases of iNOS expression and NO levels and reversed TNF-alpha-induced decreases of sGCalpha1, sGCbeta1, and PKG expression. These results imply that cGMP enhancers could have anti-proinflammatory potential in TSMCs. TNF-alpha also increased protein kinase A (PKA) expression and cAMP levels, cyclooxygenase-2 (COX-2) expression, and activated productions of prostaglandin (PG) E2 and 6-keto-PGF1alpha (stable PGI2 metabolite). Dexamethasone and N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide (NS-398; a selective COX-2 inhibitor) attenuated TNF-alpha-induced expression of COX-2 and activated productions PGE2 and PGI2. However, KMUP-1 and KMUP-3 did not affect COX-2 activities and did not further enhance cAMP levels in the presence of TNF-alpha. It is suggested that TNF-alpha-induced increases of PKA expression and cAMP levels are mediated by releasing PGE2 and PGI2, the activation products of COX-2. In conclusion, xanthine-based KMUP-1 and KMUP-3 inhibit TNF-alpha-induced expression of iNOS in TSMCs, involving the sGC/cGMP/PKG expression pathway but without the involvement of COX-2.

Guanylate cyclase (GC) catalyzes the biosynthesis of cyclic guanosine 3',5'- monophosphate (cGMP) from GTP. GC exists in two isoenzyme forms: soluble and membrane-bound. The soluble GC (sGC) is a heterodimer composed of an alpha and a beta subunit, and it contains heme as a prosthetic group. The most important physiological activator of sGC is nitric oxide, which activates the enzyme upon binding to the heme moiety. By producing the second messenger cGMP, which regulates various effector systems such as phosphodiesterases, ion channels, and protein kinases, sGC plays an important role in different physiological processes, thus representing a very attractive pharmacological target. In fact, the pathogenesis of several diseases, especially those of the cardiovascular system, has been linked to inappropriate regulation of sGC. In order to find new modulators for this important enzyme, an innovative cell-based assay has been developed and optimized for the use in high-throughput screening. This luminescent assay, which is suitable for both 96- and 384-well plate formats, has been achieved by stably expressing the alpha and beta subunits of a mutated form of sGC in Chinese hamster ovary cells. The mutated form synthesizes cyclic adenosine 3',5'-monophosphate instead of cGMP, allowing the detection of enzymatic activity by a reporter gene approach. We demonstrated that this cell line responds to compounds typically used in the field of sGC research and that it represents an innovative and robust assay to screen for sGC modulators with high efficiency and high sensitivity by means of standard luminescence readers.

Soluble guanylate cyclases (s GC s) are eukaryotic heme sensor proteins that selectively bind NO in the presence of a large excess of the similar diatomic gas, O(2); this discrimination is essential for NO signaling. Recent discoveries place sGC in the H-NOX (heme nitric oxide and/or oxygen binding domain) family that includes bacterial proteins. The defining characteristic of this family is that some H-NOX proteins tightly bind O(2) whereas others, such as sGC, show no measurable affinity for O(2). A molecular basis for this ligand selectivity has now been established. A distal pocket tyrosine is requisite for O(2) binding and is used to kinetically distinguish between NO and O(2). In the absence of this tyrosine, the O(2) dissociation rate is so fast that the O(2) complex is never formed, whereas the rate of NO dissociation remains essentially unchanged, thus providing discrimination.

Summary 1 Our aim was to study the role of nitric oxide (NO) and arachidonic acid pathways in the α1-adrenoceptor-mediated vasoconstriction in mesenteric resistance arteries from 3–4 and 22 to 23-month-old Sprague-Dawley rats. 2 The expression of NO synthase (NOS), cyclooxygenase (COX) isoforms, soluble guanylate cyclase, superoxide dismutase and the NAD(P)H oxidase subunits p22phox and p47phox were determined. 3 The NG-nitro-l-arginine methyl ester, a non-selective NOS inhibitor, shifted to the left but indomethacin and NS 398, non-selective and selective COX-2 inhibitors, shifted to the right the concentration-response curve for the vasoconstriction by phenylephrine in both age groups. 4 Ageing up-regulated endothelial NOS and p22phox expression but did not modify COX, soluble guanylate cyclase, superoxide dismutase and p47phox expression. 5 These data suggest that the observed enhancement of eNOS protein expression could constitute a compensatory mechanism to counter-regulate a chronic loss of NO possibly through increased superoxide anion production from NAD(P)H oxidase induced by age.

Background: Cyclic GMP (cGMP) concentration is increased in plasma of patients with liver cirrhosis. Three possible mechanisms may contribute: increased cGMP synthesis by soluble (activated by nitric oxide), or particulate (activated by atrial natriuretic peptide (ANP)) guanylate cyclase or increased release from cells. Aim: The aim of this work was to analyze the possible contributors to increased plasma cGMP and to assess whether changes in the parameters of the system vary with the degree of liver disease (Child Pugh score) or by the presence of ascites. Methods: We measured cGMP in plasma and lymphocytes, soluble guanylate cyclase activation by nitric oxide in lymphocytes, nitrates and nitrites and ANPs (activator of particulate guanylate cyclase) in plasma. We analyzed the correlation between changes in different parameters to discern which parameters contribute to increased plasma cGMP. Results: The plasma content of nitrates+nitrites, ANP and cGMP are increased. Activation of soluble guanylate cyclase by nitric oxide is increased in patients while basal cGMP in lymphocytes is decreased. Conclusions: Both increased ANP and increased activation of soluble guanylate cyclase by nitric oxide contribute to increased plasma cGMP in patients. The concentrations of ANP and cGMP in plasma increase with the degree of disease and are higher in patients with ascites.

Soluble guanylate cyclase, a heterodimer consisting of an alpha- and a heme-containing beta-subunit, is the major receptor for the biological messenger nitric oxide (NO) and is involved in various signal transduction pathways. The heme moiety of the enzyme is bound between the axial heme ligand histidine(105) and the recently identified counterparts of the heme propionic acids, tyrosine(135) and arginine(139). The latter residues together with an invariant serine(137) form the unique heme binding motif Y-x-S-x-R. In this work, we show that replacement of the serine(137) with alanine destabilizes the binding of the heme moiety and impairs NO-mediated soluble guanylate cyclase activation.

We have established a rapid, homogeneous, cell-based, and highly sensitive assay for guanosine 3'-5'-cyclic monophosphate (cGMP) that is suitable for fully automated ultra-high-throughput screening. In this assay system, cGMP production is monitored in living cells via Ca2+ influx through the olfactory cyclic nucleotide-gated cation channel CNGA2, acting as the intracellular cGMP sensor. A stably transfected Chinese hamster ovary (CHO) cell line was generated recombinantly expressing soluble guanylate cyclase, CNGA2, and aequorin as a luminescence indicator for the intracellular calcium concentration. This cell line was used to screen more than 900,000 compounds in an automated ultra-high-throughput screening assay using 1536-well microtiter plates. In this way, we have been able to identify BAY 58-2667, a member of a new class of amino dicarboxylic acids that directly activate soluble guanylate cyclase. The assay system allows the real-time cGMP detection within living cells and makes it possible to screen for activators and inhibitors of enzymes involved in the nitric oxide/cGMP pathway.

Adaptation to hypoxia and maintenance of O(2) homeostasis involve a wide range of responses that occur at different organizational levels in the body. One of the most important transcription factors that activate the expression of O(2)-regulated genes is hypoxia-inducible factor 1 (HIF-1). Nitric oxide (NO) mediates a variety of biological effects including relaxation of blood vessels and cytotoxicity of activated macrophages. We investigated the effect of the clinically used nitrates nitroglycerin (NTG), isosorbide dinitrate (ISDN), and sodium nitroprusside (SNP) on HIF-1-mediated transcriptional responses to hypoxia. We demonstrate that among the three nitrates, only SNP inhibits HIF-1 activation in response to hypoxia. In contrast, NTG or ISDN does not affect HIF-1 activity. SNP inhibits the accumulation of HIF-1alpha, the regulatory subunit of HIF-1, and the transcriptional activation of HIF-1alpha via a mechanism that is not dependent on either NO or soluble guanylate cyclase.

The natriuretic peptide receptor guanylate cyclases are members of the membrane-bound guanylate cyclase family. Atrial natriuretic pepticle (ANP) stimulates guanylate cyclase A, which is predominantly expressed in the rat parotid gland. ATP is well known to increase ANP-stimulated guanylate cyclase, with Mg2+ as a cofactor. We investigated the regulation of guanylate cyclase activity in rat parotid membranes with Mn2+ as a cofactor, because enzyme activity was much higher with Mn2+ than with Mg2+. ANP (10(-7)M) stimulated guanylate cyclase activity to 120%-130% of the control level, and ATP (0.1-1.0 mM) depressed its activity, with or without ANP, to 70%-80% of the control. Enzyme activity was increased by the addition of phosphate (5-20 mM). In the presence of phosphate (5 mM), guanylate cyclase with and without ANP was maximally stimulated to 5- and 6.6-fold of the control, respectively. The net stimulation of guanylate cyclase by ANP was increased at ATP concentrations between 0.2 and 0.5 mM in the presence of phosphate (5 mM or 10 mM), but no change was observed in the absence of phosphate. Phosphate not only stimulated guanylate cyclase in the absence of ATP but altered the ATP regulation of ANP-stimulated guanylate cyclase. The stimulation of guanylate cyclase by phosphate may depend on a peculiarity of rat parotid membranes.

Soluble guanylate cyclase (sGC) is a nitric oxide- (NO-) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. Characterization of two of these proteins is reported here. The first is a 181 amino acid protein cloned from Vibrio cholerae (VCA0720) that is encoded in a histidine kinase-containing operon. The ferrous unligated form of VCA0720 is 5-coordinate, high-spin. The CO complex is low-spin, 6-coordinate, and the NO complex is high-spin and 5-coordinate. These ligand-binding properties are very similar to those of sGC. The second protein is the N-terminal 188 amino acids of Tar4 (TtTar4H), a predicted methyl-accepting chemotaxis protein (MCP) from the strict anaerobe Thermoanaerobacter tengcongensis. TtTar4H forms a low-spin, 6-coordinate ferrous-oxy complex, the first of this sGC-related family that binds O2. TtTar4H has ligand-binding properties similar to those of the heme-containing O2 sensors such as AxPDEA1. sGC does not bind O2 despite having a porphyrin with a histidyl ligand like the globins. The results reported here, with sequence-related proteins from prokaryotes but in the same family as the sGC heme domain, show that these proteins have evolved to discriminate between ligands such as NO and O2; hence, we term this family H-NOX domains (heme-nitric oxide/oxygen).

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.

GC-C (guanylate cyclase C) is the receptor for heat-stable enterotoxins, guanylin and uroguanylin peptides. Ligand binding to the extracellular domain of GC-C activates the guanylate cyclase domain leading to accumulation of cGMP. GC-C is expressed as differentially glycosylated forms in HEK-293 cells (human embryonic kidney-293 cells). In the present study, we show that the 145 kDa form of GC-C contains sialic acid and galactose residues and is present on the PM (plasma membrane) of cells, whereas the 130 kDa form is a high mannose form that is resident in the endoplasmic reticulum and serves as the precursor for the PM-associated form. Ligand-binding affinities of the differentially glycosylated forms are similar, indicating that glycosylation of GC-C does not play a role in direct ligand interaction. However, ligand-stimulated guanylate cyclase activity was observed only for the fully mature form of the receptor present on the PM, suggesting that glycosylation had a role to play in imparting a conformation to the receptor that allows ligand stimulation. Treatment of cells at 20 °C led to intracellular accumulation of a mature glycosylated form of GC-C that now showed ligand-stimulated guanylate cyclase activity, indicating that localization of GC-C was not critical for its catalytic activity. To determine if complex glycosylation was required for ligand-stimulated activation of GC-C, the receptor was expressed in HEK-293 cells that were deficient in N-acetylglucosaminyltransferase 1. This minimally glycosylated form of the receptor was expressed on the cell surface and could bind a ligand with an affinity comparable with the 145 kDa form of the receptor. However, this form of the receptor was poorly activated by the ligand. Therefore our studies indicate a novel role for glycosidic modification of GC-C during its biosynthesis, in imparting subtle conformational changes in the receptor that allow for ligand-mediated activation and perhaps regulation of basal activity.

Natriuretic peptides (NP) activate particulate guanylate cyclase (pGC) and nitric oxide (NO) activates soluble guanylate cyclase (sGC). Both guanylate cyclases catalyse the formation of the same second messenger, cyclic guanosine 3',5'-monophosphate (cGMP), which activates the cGMP-dependent protein kinases (PKG). PKG then starts a signalling cascade that mediates many cardiovascular and renal effects, such as smooth muscle relaxation and diuresis. Many cell types possess both sGC and pGC. Because both GC-cGMP systems play complementary roles, an interaction between the two pathways might represent an important physiological control mechanism. In this report we demonstrate an interaction between the two pathways. C-type natriuretic peptide (CNP) decreased the beta-subunit of sGC (sGC-beta) steady-state protein levels and enzymatic activity in cultured human mesangial cells (HMC) in a time- and dose-dependent manner. This down-regulation was not dependent on changes in sGC-beta mRNA levels. Treatment of the cells with the stable cGMP analogue 8-Br-cGMP or the phosphodiesterase type-5 inhibitor Zaprinast produced the same down-regulatory effect. Inhibition of PKG or proteasome activity prevented the CNP-induced reduction of sGC-beta protein levels and activity. Taken together, these results demonstrate that pGC activation induces a post-transductional down-regulation of sGC by a mechanism involving PKG and the proteasome pathway.

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.

Although nitric oxide (NO) was shown not only to exert biological activities through activation of soluble guanylate cyclase (sGC), but also to cause oxidative stress, mechanisms for switching these pathways are unknown. This study aimed to examine aberrant utilization of NO under disease conditions such as diabetes mellitus. Diabetes was induced in male Wistar rats by injecting streptozotocin (STZ; 50 mg/kg body weight, i.p.). Retina was perfusion-fixed for immunohistochemistry to detect the gas-mediated activation of sGC by anti-sGC antibodies that are function-sensitive [monoclonal antibody (MoAb) 3221] and -insensitive (MoAb28131). Regional lipid peroxidation was also examined by an anti-acrolein MoAb. At 6 weeks after STZ injection, inducible NO synthase induction became evident, coinciding with the overproduction of nitrotyrosine, followed by that of acrolein. Despite such NO overproduction, sGC did not exhibit any notable activation. When STZ-treated animals were posttreated with a derivative of superoxide dismutase that stays in circulation without undergoing renal ultrafiltration, immunoreactivities to MoAb3221 but not to MoAb28131 increased markedly in diabetic retina, suggesting that superoxide cancels free NO for local sGC activation. These results provide evidence of aberrant utilization of NO and suggest that superoxide plays a role in interfering with NO-mediated sGC activation for phototransducing events in this neural tissue.

Cyclic guanosine-3',5'-monophosphate (cGMP)-mediated mechanisms play an important role in vasodilation and blood pressure regulation. We investigated basal activity of the nitric oxide (NO)-cGMP signal transduction pathway in corpus cavernosum from both middle-aged and young rats, and the electrical field stimulation-induced relaxation in the organ was also evaluated. In middle-aged rats, nitric oxide synthase (NOS) and cGMP-phosphodiesterase activities were significantly decreased; however, guanylate cyclase activity was similar. cGMP concentration, a secondary messenger of NO, remained almost the same level as compared with young rats. These results suggest that decrease in cGMP-phosphodiesterase activity is likely to account for the maintenance of cGMP concentration. In isolated corpus cavernosum from middle-aged rats, electrical field stimulation-induced relaxation was partially impaired. These results suggest that downregulation of the NOS and cGMP-phosphodiesterase activities are early events in the pathogenesis of erectile dysfunction.

Accumulated evidence suggests that quercetin, a dietary flavonoid, has beneficial effects in protection against cardiovascular diseases and in the inhibition of tumour growth. We have recently shown that antioxidants such as 17β-estradiol, resveratrol, dithiothreitol and vitamin C activate membrane-bound guanylate cyclase GC-A, a receptor for atrial natriuretic factor (ANF). Since quercetin is a phytoestrogen and potent antioxidant, it is possible that it may activate GC-A or other guanylate cyclase isoforms. We examined whether quercetin activates GC-A or GC-B (the receptor for C-type natriuretic peptide, CNP) in PC12 and porcine kidney proximal tubular LLC-PK1 cells. The results showed that quercetin activated a guanylate cyclase isoform in both cell types. Quercetin inhibited CNP-stimulated GC-B activity, but had little effect on ANF-stimulated GC-A activity in PC12 cells, suggesting that quercetin mainly activates GC-B in PC12 cells. In contrast, CNP had no effect on guanylate cyclase activity in LLC-PK1 cells, indicating that GC-B is not expressed in LLC-PK1 cells. Furthermore, quercetin had a small effect on ANF-stimulated GC-A activity and had no effect on soluble guanylate cyclase activity in LLC-PK1 cells, suggesting that quercetin does not activate GC-A, GC-B or soluble guanylate cyclase in LLC-PK1 cells. However, quercetin did stimulate membrane-bound guanylate cyclase activity in LLC-PK1 cell membranes. These results indicate that quercetin activates the GC-B isoform in PC12 cells, but activates an unknown membrane-bound guanylate cyclase isoform in LLC-PK1 cells.

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.