Soluble Guanylyl Cyclase Subunits Research Articles

Loss of Gucy1a3 causes poor post-stroke recovery by reducing angiogenesis via the HIF-1α/VEGFA signaling pathway in mice

ObjectivesIschemic stroke is a common and debilitating disease that can cause permanent neurological damage. Gucy1a3, which encodes the α1 subunit of soluble guanylyl cyclase, has been reported to be associated with functional recovery after ischemic stroke. However, the mechanism is still not well understood. In the present study, we investigated the effects of Gucy1a3 on (i) post-stroke recovery; (ii) vascular endothelial growth factor A (VEGFA) and hypoxia inducible factor 1 alpha (HIF-1α) expression; and (iii) angiogenesis after ischemic stroke. Materials and methodsWild-type and Gucy1a3 knockout C57BL/6J male mice were respectively used to establish the models of permanent middle cerebral artery occlusion (pMCAO). Neurological deficit scores were evaluated at 24 h and 96 h after pMCAO. Cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. For determining microvessel density, immunohistochemical analysis was performed with CD31. The expression of VEGFA and HIF-1α was detected by western blotting. ResultsOur results suggest that loss of Gucy1a3 increased the infarct volume and aggravated neurological deficits after pMCAO. In addition, the Gucy1a3 knockout brains exhibited significantly lower microvessel densities and VEGFA and HIF-1α expression levels than the wild-type brains at 96 h post-pMCAO. ConclusionsOur study indicates that GUCY1A3 might be involved in angiogenesis after ischemic stroke. Further investigation of GUCY1A3 will provide a new therapeutic target for stroke.

Co-expression of soluble guanylyl cyclase subunits and PDE5A shRNA to elevate cellular cGMP level: A potential gene therapy for myocardial cell death.

Genetic manipulation on the NO-sGC-cGMP pathway has been rarely achieved, partially due to complexity of the soluble guanylyl cyclase (sGC) enzyme. We aim to develop gene therapy directly targeting the pathway to circumvent cytotoxicity and tolerance after prolonged use of NO-donors and the insufficiency of PDE inhibitors. In this study, we constructed lentivirus vectors expressing GUCY1A3 and GUCY1B3 genes, which encoded the α1 and β1 subunits of soluble guanylyl cyclase (sGC), respectively, to enhance cGMP synthesis. We also constructed lentiviral vector harboring PDE5A shRNA to alleviate phosphodiesterase activity and cGMP degradation. Transductions of human HEK293 cells with the constructs were successful, as indicated by the fluorescent signal and altered gene expression produced by each vector. Overexpression of GUCY1A3 and GUCY1B3 resulted in increased sGC enzyme activity and elevated cGMP level in the cells. Expression of PDE5A shRNA resulted in decreased PDE5A expression and elevated cGMP level. Co-transduction of the three lentiviral vectors resulted in a more significant elevation of cGMP in HEK293 cells without obvious cytotoxicity. To the best of our knowledge, this is the first study to show that co-expression of exogenous subunits of the soluble guanylyl cyclase could form functional enzyme and increase cellular cGMP level in mammalian cells. Simultaneous expression of PDE5A shRNA could alleviate feedback up-regulation on PDE5A caused by cGMP elevation. Further studies are required to evaluate the effects of these constructs in vivo.

Dental Pulp Inflammation Initiates the Occurrence of Mast Cells Expressing the α1 and β1 Subunits of Soluble Guanylyl Cyclase.

The binding of nitric oxide (NO) to heme in the β1 subunit of soluble guanylyl cyclase (sGC) activates both the heterodimeric α1β1 and α2β1 isoforms of the enzyme, leading to the increased production of cGMP from GTP. In cultured human mast cells, exogenous NO is able to inhibit mast cell degranulation via NO-cGMP signaling. However, under inflammatory oxidative or nitrosative stress, sGC becomes insensitive to NO. The occurrence of mast cells in healthy and inflamed human tissues and the in vivo expression of the α1 and β1 subunits of sGC in human mast cells during inflammation remain largely unresolved and were investigated here. Using peroxidase and double immunohistochemical incubations, no mast cells were found in healthy dental pulp, whereas the inflammation of dental pulp initiated the occurrence of several mast cells expressing the α1 and β1 subunits of sGC. Since inflammation-induced oxidative and nitrosative stress oxidizes Fe2+ to Fe3+ in the β1 subunit of sGC, leading to the desensitization of sGC to NO, we hypothesize that the NO- and heme-independent pharmacological activation of sGC in mast cells may be considered as a regulatory strategy for mast cell functions in inflamed human dental pulp.

Nitric oxide-soluble guanylyl cyclase pathway as a contributor to age-related memory impairment in Drosophila.

Age‐related changes in the transcriptome lead to memory impairment. Several genes have been identified to cause age‐dependent memory impairment (AMI) by changes in their expression, but genetic screens to identify genes critical for AMI have not been performed. The fruit fly is a useful model for studying AMI due to its short lifespan and the availability of consistent techniques and environments to assess its memory ability. We generated a list of candidate genes that act as AMI regulators by performing a comprehensive analysis of RNAsequencing data from young and aged fly heads and genome‐wide RNAi screening data to identify memory‐regulating genes. A candidate screen using temporal and panneuronal RNAi expression was performed to identify genes critical for AMI. We identified the guanylyl cyclase β‐subunit at 100B (gycβ) gene, which encodes a subunit of soluble guanylyl cyclase (sGC), the only intracellular nitric oxide (NO) receptor in fruit flies, as a negative regulator of AMI. RNAi knockdown of gycβ in neurons and NO synthase (NOS) in glia or neurons enhanced the performance of intermediate‐term memory (ITM) without apparent effects on memory acquisition. We also showed that pharmacological inhibition of sGC and NOS enhanced ITM in aged individuals, suggesting the possibility that age‐related enhancement of the NO‐sGC pathway causes memory impairment.

Involvement of nNOS, and α1, α2, β1, and β2 Subunits of Soluble Guanylyl Cyclase Genes Expression in Anticonvulsant Effect of Sumatriptan on Pentylenetetrazole-Induced Seizure in Mice.

Epileptic seizure is phenomenon of abnormal synchronous neuronal discharge of a set of neurons in brain as a result of neuronal excitation. Evidence shows the nitric oxide (NO) involvement in neuronal excitability. Moreover, the role of cyclic guanosine monophosphate (cGMP) activation in seizure pathogenesis is well-established. Sumatriptan is a selective agonist of 5-Hydroxytryptamine1B/D auto-receptor, has been reassessed for its neuroprotection. This study was aimed to explore the anticonvulsant effect of sumatriptan through possible involvement of NO-cGMP pathway in mice. For this purpose, the protective effect of sumatriptan on PTZ-induced clonic seizure threshold (CST) was measured using NO-cGMP pathway inhibitors including N(G)-nitro-L-arginine (L-NNA, 1, 5, and 10 mg/kg), 7-nitroindazole (7-NI, 30, 45, and 60 mg/kg), aminoguanidine (AG, 30, 50, and 100 mg/kg), methylene blue (MB, 0.1, 0.5, and 1 mg/kg) and sildenafil (5, 10, and 20 mg/kg). The involvement of nitrergic system was further confirmed by measurement of nitrite levels by Griess reaction. The gene expression of neuronal nitric oxide synthase (nNOS) and subunits of soluble guanylyl cyclase (sGC) was studied using qRT-PCR analysis. Acute administration of sumatriptan (1.2 and 0.3 mg/kg) in combination with subeffective doses of NOS, sGC, and phosphodiesterase 5 inhibitors significantly reversed the PTZ-induced CST (P ≤ 0.001). The nitrite level in prefrontal cortex was significantly attenuated by sumatriptan (P ≤ 0.01). Furthermore, sumatriptan downregulated the PTZ-induced mRNA expression of nNOS (P ≤ 0.01), α1 (P ≤ 0.001), α2 (P ≤ 0.05), and β1 (P ≤ 0.05) genes in cerebral cortex of mice. In conclusion, the anticonvulsant activity of sumatriptan at least, in part, is mediated through inhibiting NO-cGMP pathway.

TMPRSS2-ERG activates NO-cGMP signaling in prostate cancer cells

The aberrant activation of the ERG oncogenic pathway due to the TMPRSS2-ERG gene fusion is the major event that contributes to prostate cancer (PCa) development. However, the critical downstream effectors that can be therapeutically targeted remain to be identified. In this study, we have found that the expression of the α1 and β1 subunits of soluble guanylyl cyclase (sGC) was directly and specifically regulated by ERG in vitro and in vivo and was significantly associated with TMPRSS2-ERG fusion in clinical PCa cohorts. sGC is the major mediator of nitric oxide (NO)-cGMP signaling in cells that, upon NO binding, catalyzes the synthesis of cGMP and subsequently activates protein kinase G (PKG). We showed that cGMP synthesis was significantly elevated by ERG in PCa cells, leading to increased PKG activity and cell proliferation. Importantly, we also demonstrated that sGC inhibitor treatment repressed tumor growth in TMPRSS2-ERG-positive PCa xenograft models and can act in synergy with a potent AR antagonist, enzalutamide. This study strongly suggests that targeting NO-cGMP signaling pathways may be a novel therapeutic strategy to treat PCa with TMPRSS2-ERG gene fusion.

Exercise training induces eNOS coupling and restores relaxation in coronary arteries of heart failure rats.

Exercise training (ET) has emerged as a nonpharmacological therapy for cardiovascular diseases because of its helpful milieu for improving vascular function. The aim of the present study was to assess whether ET reverses the alterations in vascular reactivity observed in heart failure (HF)-related coronary arteries and to elucidate the molecular mechanisms involved in these adjustments. Male Wistar rats were subjected to either coronary artery ligation or sham operation. Four weeks after the surgery, rats were divided into two groups: untrained HF (UHF) and exercise-trained HF (THF). ET was conducted on a treadmill for 8 wk. An untrained SO group was included in the study as a normal control. ET restored the impaired acetylcholine (ACh)- and sodium nitroprusside-induced relaxation in coronary arteries to levels of the control. Oxidative stress and reduced nitric oxide (NO) production were observed in UHF, whereas ET restored both parameters to the levels of the control. Expression levels of endothelial NO synthase (eNOS) and soluble guanylyl cyclase subunits were increased in coronary arteries of UHF rats but reduced in THF rats. Tetrahydrobiopterin restored ACh-induced NO production in the UHF group, indicating that eNOS was uncoupled. ET increased the eNOS dimer-to-monomer ratio and expression of GTP cyclohydrolase 1, thus increasing NO bioavailability. Taken together, these findings demonstrate that ET reverses the dysfunction of the NO/soluble guanylyl cyclase pathway present in coronary arteries of HF rats. These effects of ET are associated with increased GTP cyclohydrolase 1 expression, restoration of NO bioavailability, and reduced oxidative stress through eNOS coupling. NEW & NOTEWORTHY The present study provides a molecular basis for the exercise-induced improvement in coronary arteries function in heart failure. Increasing the expression of GTP cyclohydrolase 1, the rate-limiting enzyme in the de novo biosynthesis of tetrahydrobiopterin, exercise training couples endothelial nitric oxide synthase, reduces oxidative stress, and increases nitric oxide bioavailability and sensitivity in coronary arteries of heart failure rats.

Hypertension reduces soluble guanylyl cyclase expression in the mouse aorta via the Notch signaling pathway

Hypertension is a dominating risk factor for cardiovascular disease. To characterize the genomic response to hypertension, we administered vehicle or angiotensin II to mice and performed gene expression analyses. AngII treatment resulted in a robust increase in blood pressure and altered expression of 235 genes in the aorta, including Gucy1a3 and Gucy1b3 which encode subunits of soluble guanylyl cyclase (sGC). Western blotting and immunohistochemistry confirmed repression of sGC associated with curtailed relaxation via sGC activation. Analysis of transcription factor binding motifs in promoters of differentially expressed genes identified enrichment of motifs for RBPJ, a component of the Notch signaling pathway, and the Notch coactivators FRYL and MAML2 were reduced. Gain and loss of function experiments demonstrated that JAG/NOTCH signaling controls sGC expression together with MAML2 and FRYL. Reduced expression of sGC, correlating with differential expression of MAML2, in stroke prone and spontaneously hypertensive rats was also seen, and RNA-Seq data demonstrated correlations between JAG1, NOTCH3, MAML2 and FRYL and the sGC subunits GUCY1A3 and GUCY1B3 in human coronary artery. Notch signaling thus provides a constitutive drive on expression of the major nitric oxide receptor (GUCY1A3/GUCY1B3) in arteries from mice, rats, and humans, and this control mechanism is disturbed in hypertension.

Neuronal nitric oxide synthase localizes to utrophin expressing intercalated discs and stabilizes their structural integrity

The neuronal nitric-oxide synthase (nNOS) splice variant nNOSµ is essential for skeletal muscle function. Its localization is dependent on dystrophin, which stabilizes the dystrophin glycoprotein complex (DGC) at the sarcolemma of skeletal muscle fibers. In Duchenne muscular dystrophy (DMD) dystrophin is absent and sarcolemmal nNOS is lost. This leads to functional ischemia due to a decrease in contraction-induced vasodilation. In cardiomyocytes, nNOSµ is believed to be the predominant NOS isoform. However, the association of nNOS with the DGC in the heart is unclear. Here, we report nNOS localization at the intercalated discs (IDs) of cardiomyocytes, where utrophin is highly expressed. In mdx, mdx:utr, nNOSµ knock-out (KO), and mdx:nNOSµ KO mice, we observed a gradual reduction of nNOS at IDs and disrupted ID morphology, compared to wild-type. In mdx:nNOSµ KO mice, but not in mdx or nNOSµ KO mice, we also observed an early development of cardiac fibrosis. These findings suggest that nNOS localization in the heart may not depend exclusively on the presence of dystrophin. Additionally, the β1 subunit of soluble guanylyl cyclase (sGC), responsible for the production of cGMP through nitric oxide (NO) signaling, was also detected at the IDs. Together, our results suggest a new role of nNOS at the IDs for the cGMP-dependent NO pathway and the maintenance of ID morphology.

YC-1 Binding to the β Subunit of Soluble Guanylyl Cyclase Overcomes Allosteric Inhibition by the α Subunit

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remain unknown. Using linked equilibrium binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the β subunit. In the absence of CO, YC-1 binds with a Kd of 9-21 μM, depending on the construct. In the presence of CO, these values decrease to 0.6-1.1 μM. Pfizer compound 25 bound ∼10-fold weaker than YC-1 in the absence of CO, whereas compound BAY 41-2272 bound particularly tightly in the presence of CO (Kd = 30-90 nM). Additionally, we found that CO binds much more weakly to heterodimeric sGC proteins (Kd = 50-100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca β H-NOX/PAS). YC-1 greatly enhanced binding of CO to heterodimeric sGC, as expected (Kd ∼ 1 μM). These data indicate the α subunit induces a heme pocket conformation with a lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the α subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity.

Dysfunctional nitric oxide signalling increases risk of myocardial infarction

Myocardial infarction, a leading cause of death in the Western world, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the α1 subunit of soluble guanylyl cyclase (α1-sGC), and CCT7 encodes CCTη, a member of the tailless complex polypeptide 1 ring complex, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce α1-sGC as well as β1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in α1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction.

Decrease of guanylyl cyclase β1 subunit and nitric oxide (NO)-induced relaxation in mouse rectum with colitis and its reproduction on long-term NO treatment

Nitric oxide (NO) influences motility in the colon in patients with ulcerative colitis, but the exact mechanism involved remains unknown. Colitis was induced in mice by the oral administration of 2.5% dextran sodium sulfate (DSS), and the motility in longitudinal preparations from rectum and distal colon and expression of β1 subunit of soluble guanylyl cyclase (sGCβ1) were analyzed. Electrical stimulation (ES) caused a transient relaxation via the NO pathway in both rectum and colon from control mice. Stimulation with sodium nitroprusside (SNP) caused relaxation in the two regions, and the half-time (T (1/2)) of the maximal relaxation induced by 100μM SNP was 8.1 ± 1.0s in rectum. DSS treatment (1) abolished the ES-induced relaxation, but not dibutyryl cyclic GMP-induced response, in both regions, (2) decreased the maximal response to SNP accompanied by a loss of immunoreactive sGCβ1 protein in rectum, but did not affect the amplitude of the relaxant response or the protein in distal colon, and (3) caused an increase in the T (1/2) value in response to SNP in both regions. Pretreatment of both preparations from control mice with 600μM SNP for 30min decreased both ES- and SNP-induced relaxation, SNP-induced cyclic GMP formation, and immunoreactive sGCβ1 levels. NO-mediated relaxation was impaired by a dysfunctional sGC with and without a loss of immunoreactivity to sGCβ1 in rectum and colon from DSS-treated mice, respectively. Long-term exposure of the tissues with an excess amount of NO changes the sGC-mediated relaxation.

Notch Initiates the Endothelial-to-Mesenchymal Transition in the Atrioventricular Canal through Autocrine Activation of Soluble Guanylyl Cyclase

The heart is the most common site of congenital defects, and valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn. The process of endothelial-to-mesenchymal transition (EndMT) in the cardiac cushions is a required step during early valve development, and Notch signaling is required for this process. Here we show that Notch activation induces the transcription of both subunits of the soluble guanylyl cyclase (sGC) heterodimer, GUCY1A3 and GUCY1B3, which form the nitric oxide receptor. In parallel, Notch also promotes nitric oxide (NO) production by inducing Activin A, thereby activating a PI3-kinase/Akt pathway to phosphorylate eNOS. We thus show that the activation of sGC by NO through a Notch-dependent autocrine loop is necessary to drive early EndMT in the developing atrioventricular canal (AVC).

Identification of NO/sGC signalling in the bulbospinal respiratory pathway after C2–C3 hemisection of the spinal cord in rats

Guanylyl cyclase (GC) as the effector molecule for nitric oxide (NO) plays a key role in the NO/cGMP signalling cascade. Based on these observations, our study focused on NO/sGC signalization in the bulbospinal respiratory pathway. The distribution of neuronal nitric oxide synthase (nNOS), β1 subunit of soluble guanylyl cyclase (β1sGC) and synaptophysin (SYN) was explored in the upper part of the respiratory pathway after C2–C3 hemisection of the spinal cord in male Wistar rats. Unilateral injection of Fluorogold into the phrenic nucleus (PN) at C4 level and survival of animals for 2 days revealed many Fluorogold fluorescent neurons in the ventral respiratory group (VRG) of the medulla, mostly on the contralateral side. Under physiological conditions we noted nNOS-fluorescent terminals of VRG neurons around β1sGC fluorescent motoneurons in the PN. A strong depletion of nNOS/SYN fluorescent terminals was noted 8 days after hemisection around alpha motoneurons in the PN on the contralateral side. On the side of injury, nNOS/SYN fluorescent puncta were detected around phrenic motoneurons only sporadically. Phrenic alpha motoneurons responded to C2–C3 hemisection by a loss of β1sGC positivity. The results confirm, that β1sGC immunoreactive phrenic motoneurons are innervated by nNOS positive terminals coming from the VRG.

Is Nostoc H-NOX a NO Sensor or Redox Switch?

Nostoc sp. (Ns) H-NOX is a heme protein found in symbiotic cyanobacteria, which has approximately 35% sequence identity and high structural homology to the beta subunit of soluble guanylyl cyclase (sGC), suggesting a NO sensing function. However, UV-vis, EPR, NIR MCD, and ligand binding experiments with ferrous and ferric Ns H-NOX indicate significant functional differences between Ns H-NOX and sGC. (1) After NO binding to sGC, the proximal histidine dissociates from the heme iron, causing a conformational change that triggers activation of sGC. In contrast, formation of pentacoordinate (5c) NO heme occurs to only a limited extent in Ns H-NOX, even at >1 mM NO. (2) Unlike sGC, two different hexacoordinate (6c) NO complexes are formed in Ns H-NOX with initial and final absorbance peaks at 418 and 414 nm, and the conversion rate is linearly dependent on [NO], indicating that a second NO binds transiently to catalyze formation of the 414 nm species. (3) sGC is insensitive to oxygen, and ferric sGC prepared by ferricyanide oxidation has a 5c high-spin heme complex. In contrast, Ns H-NOX autoxidizes in 24 h if exposed to air and forms a 6c ferric heme complex, indicating a major conformational change after oxidation and coordination by a second histidine side chain. Such a large conformational transition suggests that Ns H-NOX could function as either a redox or a NO sensor in the cyanobacterium.

Selective activation of hippocampal guanylyl cyclase by a novel NO mimetic nitrate ester

Studies of the distribution of α1 and α2 subunits of soluble guanylyl cyclase (sGC) indicate that the α2 subunit is expressed in relatively high abundance in brain, and is the major alpha subunit present in the hippocampus. Furthermore, the α2 subunit interacts with a PDZ domain of PSD95, a scaffolding protein that couples the NMDA receptor to neuronal nitric oxide synthase. Thus a membrane-bound NO-sensitive sGC is present in the hippocampus, and has been suggested to be a component of a signal transduction complex important for modulation of synaptic transmission and plasticity. We recently completed a study in which a novel NO mimetic nitrate ester (GT1061) reversed cognitive deficits in a cholinergic depletion model of Alzheimer's disease [1]. Of interest was that comparison of sGC activation in broken cell preparations of aorta and hippocampus showed that GT1061 selectively increased enzyme activity in the hippocampus (16to 20-fold in hippocampus vs. 2to 3 fold in aorta), suggesting that the enhanced activation of hippocampal sGC by GT1061 could be due to selectivity of the drug for targeting the α2β1 isoform of sGC.

Species differences in the localization of soluble guanylyl cyclase subunits in monkey and rat brain

Nitric oxide (NO) exerts most of its physiological effects through activation of a predominantly soluble guanylyl cyclase (sGC). In mammalian cells sGC exists as a heterodimer of alpha and beta subunits. Currently, four subunits (alpha1, alpha2, beta1, and beta2) have been characterized. We used in situ hybridization with subunit-specific 33P-labeled oligonucleotide probes to compare the anatomical distribution of sGC subunit mRNAs in rat and monkey brains. Message for all subunits except beta2 was detected in both species. The sGC subunit showing the highest expression and widest distribution was beta1. High expression for all subunits was found in basal ganglia, olfactory system, hippocampus, cortex, and cerebellum. Minor species differences in the relative distribution of alpha subunits were observed. In general, the alpha1 message was more prominent in monkey brain and the alpha2 message in rat brain. This was more evident in limbic areas and cerebellar cortex. Some differences were also observed in subunit laminar distribution in cerebral cortex. The limited species differences in sGC subunit distribution suggest that in primates, as occurs in rodents, the NO-cGMP signaling pathway will be involved in important brain functions such as memory formation, sensory processing, and behavior.

Differential Expression of Nitric Oxide Signaling Components in Undifferentiated and Differentiated Human Embryonic Stem Cells

Nitric oxide (NO) is an uncharged free-radical gas that is involved in a number of physiological and pathological events. We have examined the expression of various subunits of soluble guanylyl cyclase (sGC alpha (1), alpha (2), beta (1), beta (2)), nitric oxide synthase (s) (NOS-1, -2, -3), MLC2 (cardiac marker) and a cardiac-specific transcription factor (Nkx2.5) in human embryonic stem (hES) cells (H-9 cells) and differentiated cells subjected to differentiation in cell suspension using embryoid body (EB) formation. Our results demonstrate a time-dependent increase in the expression of sGC alpha (1) and beta (1) at the mRNA and protein levels in differentiated cells compared to undifferentiated H-9 cells as examined by real-time PCR and western blot analysis. mRNA for sGC alpha (2) also showed a time-dependent increase compared to undifferentiated cells. In contrast, there was a time-dependent decrease in sGC beta (2) mRNA expression in differentiated cells compared to undifferentiated H-9 cells. In contrast to undifferentiated H-9 cells, the maximum mRNA expression of cardiac marker MLC2 and the cardiac-specific transcription factor Nkx2.5 was observed at day 14 of the differentiated H-9 cells. The protein levels of MLC2 were stable up to day 25 compared to mRNA levels, which showed a sharp decline after day 15. Using immunofluorescence, we also demonstrate positive staining of cardiac markers such as troponin I, alpha-actinin, atrial natriuretic peptide, and SGC alpha (1) at days 8-37 post-differentiation. These results clearly demonstrate the role of NO signaling components in differentiation events or physiological processes of human ES or ES cell-derived cardiomyocytes.

Molecular cloning and characterization of α1-soluble guanylyl cyclase gene promoter in rat pituitary cells

Soluble guanylyl cyclase is a cytosolic enzyme which catalyzes conversion of GTP to the second messenger cyclic GMP. The transcriptional regulation at the promoter levels of four soluble guanylyl cyclase subunits, termed alpha1, alpha2, beta1, and beta2, is largely unknown. In this study, we identified the transcription start site of alpha1-soluble guanylyl cyclase gene in rat pituitary cells and cloned the 3.5 kb 5'-promoter. Sequence analysis of this TATA-less promoter revealed the presence of several putative-binding sites for transcriptional factors, including CCAAT site at -41 to -32 and Sp1 site at -34 to -24. Transfection of pituitary cells with constructs of variable lengths confirmed the relevance of different promoter regions in the control of transcriptional activity. Among them, the -49 to + 156 region was critical for basal transcriptional activity. Electrophoretic mobility shift assay using nuclear proteins extracted from normal and immortalized pituitary cells indicated that the CCAAT/Sp1 site within the -49 to + 156 region was able to specifically interact with CCAAT-binding factor and Sp1. These two sites were partly overlapped and both of them conferred stimulatory effects. The in vivo recruitment of CCAAT-binding factor and Sp1 was confirmed by chromatin immunoprecipitation. These results indicate that the composite CCAAT/Sp1 cis-element contributes to the expression of alpha1-sGC subunit in resting pituitary cells.

Comparison of the properties of the five soluble guanylyl cyclase subunits in Drosophila melanogaster

The Drosophila melanogaster genome contains 5 genes that code for soluble guanylyl cyclase subunits. Two of these genes code for subunits, Gycα-99B and Gycβ-100B, which form a conventional NO-sensitive guanylyl cyclase and the other three code for atypical subunits, Gyc-88E, Gyc-89Da and Gyc-89Db. The properties and distribution of Gyc-88E and Gyc-89Db have previously been described and here Gyc-89Da is described. Gyc-89Da only forms an active guanylyl cyclase when co-expressed with Gyc-88E. The three atypical subunits probably form two different heterodimers in vivo: Gyc-88E/89Da and Gyc-88E/89Db. Both of these heterodimers were slightly stimulated by NO donors and Gyc-88E/89Da showed a greater activation by Mn2 , with an increase in Vmax and a decrease in Km, compared to Gyc-88E/89Db. Both Gyc-88E/89Da and Gyc-88E/89Db were expressed in neurons in both the peripheral and central nervous system. Although all three heterodimeric soluble guanylyl cyclases in D. melanogaster can be activated by NO and inhibited by ODQ, the atypical enzymes can be distinguished from the conventional soluble guanylyl cyclase by their sensitivity to the NO-independent activators YC-1 and BAY 41-2272, which will only activate the conventional enzyme.ORF open reading frame UTR untranslated region