BAY 41-2272: A stimulator of soluble guanylyl cyclase induces NO-dependent penile erection in vivo

The decreased expression of the nitric oxide (NO) receptor, soluble guanylyl cyclase (sGC), occurs in response to multiple stimuli in vivo and in cell culture and correlates with various disease states such as hypertension, inflammation, and neurodegenerative disorders. The ability to understand and modulate sGC expression and cGMP levels in any of these conditions could be a valuable therapeutic tool. We demonstrate herein that the c-Jun NH2-terminal kinase JNK II inhibitor anthra[1,9- cd]pyrazol-6(2 H)-one (SP-600125) completely blocked the decreased expression of sGCα1-subunit mRNA by nerve growth factor (NGF) in PC12 cells. Inhibitors of the ERK and p38 MAPK pathways, PD-98059 and SB-203580, had no effect. SP-600125 also inhibited the NGF-mediated decrease in the expression of sGCα1protein as well as sGC activity in PC12 cells. Other experiments revealed that decreased sGCα1mRNA expression through a cAMP-mediated pathway, using forskolin, was not blocked by SP-600125. We also demonstrate that TNF-α/IL-1β stimulation of rat fetal lung (RFL-6) fibroblast cells resulted in sGCα1mRNA inhibition, which was blocked by SP-600125. Expression of a constitutively active JNKK2-JNK1 fusion protein in RFL-6 cells caused endogenous sGCα1mRNA levels to decrease, while a constitutively active ERK2 protein had no effect. Collectively, these data demonstrate that SP-600125 may influence the intracellular levels of the sGCα1-subunit in certain cell types and may implicate a role for c-Jun kinase in the regulation of sGCα1expression.

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Biphasic Roles for the Soluble Guanylyl Cyclase (sGC) In Platelet Activation In Mice

Blood ◽

10.1182/blood.v116.21.486.486 ◽

2010 ◽

Vol 116 (21) ◽

pp. 486-486

Author(s):

Guoying Zhang ◽

Binggang Xiang ◽

Radek C. Skoda ◽

Susan S. Smyth ◽

Xiaoping Du ◽

...

Keyword(s):

Platelet Activation ◽

Guanylyl Cyclase ◽

Conflicts Of Interest ◽

Soluble Guanylyl Cyclase ◽

Wild Type ◽

No Donor ◽

Cgmp Production ◽

Deficient Mice

Abstract Abstract 486 The role of intracellular secondary messenger cGMP in platelet activation has been controversial, with both stimulatory and inhibitory roles reported. The platelet cGMP is believed to be predominantly synthesized by soluble guanylyl cyclase (sGC), which is activated by nitric oxide (NO). To specifically determine the role of sGC-dependent cGMP synthesis in platelet function and in vivo thrombosis and hemostasis, we produced mice harboring a “floxed” sGC beta1 allele. In the “floxed” sGC beta1 mice (sGC beta1fl/fl), the exons 7 and 8 of sGC beta1 gene and an inserted Neo cassette were flanked with three LoxP sites. Platelet-specific deletion of sGC beta1fl/fl allele was accomplished through breeding of the sGC beta1fl/fl mice with pf4-Cre recombinase transgenic mice. Immunoblotting showed the complete absence of this protein in sGC beta1fl/fl/Cre platelets. Mice lacking sGC beta1 in platelets appeared to develop normally and had normal blood counts, including platelets. Blood pressure of platelet-specific sGC deficient mice was comparable to that of wild-type littermates. Inactivating the sGC beta1 gene in platelets abolished cGMP production induced by either NO donors or platelet agonists that are known to activate endogenous NO synthesis, confirming that both the platelet agonist-induced and NO donor-induced platelet cGMP production are predominantly mediated by sGC. Platelets lacking sGC exhibit a marked defect in aggregation and secretion in response to low doses of platelet agonists, collagen and thrombin. Importantly, tail-bleeding times were significantly prolonged in the platelet-specific sGC deficient mice compared with the wild-type littermates. In a FeCl3-induced carotid artery thrombosis model, time to occlusive thrombosis was prolonged in the platelet-specific sGC deficient mice, compared to wild type littermates. Thus, the agonist-stimulated sGC activation is important in promoting platelet granule secretion and aggregation. On the other hand, NO donor SNP-induced inhibition of platelet activation was abolished in sGC-deficient platelets. However, at high concentrations (>100μM), SNP inhibited platelet activation in both wild type and sGC deficient mice, indicating that both cGMP-dependent and -independent mechanisms are involved in NO donor-induced inhibition of platelet activation. Together, our data demonstrate that sGC contributes to both agonist-induced platelet activation and NO donor-induced platelet inhibition. Disclosures: No relevant conflicts of interest to declare.

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In vivo microdialysis study of a specific inhibitor of soluble guanylyl cyclase on the glutamate receptor/nitric oxide/cyclic GMP pathway

British Journal of Pharmacology ◽

10.1111/j.1476-5381.1996.tb15713.x ◽

1996 ◽

Vol 119 (3) ◽

pp. 590-594 ◽

Cited By ~ 60

Author(s):

Ernesto Fedele ◽

Ying Jin ◽

Giorgia Varnier ◽

Maurizio Raiteri

Keyword(s):

Nitric Oxide ◽

Glutamate Receptor ◽

Cyclic Gmp ◽

Guanylyl Cyclase ◽

Specific Inhibitor ◽

Soluble Guanylyl Cyclase ◽

In Vivo Microdialysis ◽

Microdialysis Study

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Penile erection requires association of soluble guanylyl cyclase with endothelial caveolin-1 in rat corpus cavernosum

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00601.2005 ◽

2006 ◽

Vol 290 (5) ◽

pp. R1302-R1308 ◽

Cited By ~ 21

Author(s):

A. Elizabeth Linder ◽

Romulo Leite ◽

Kimberly Lauria ◽

Thomas M. Mills ◽

R. Clinton Webb

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Guanylyl Cyclase ◽

Penile Erection ◽

Muscle Relaxation ◽

Corpus Cavernosum ◽

Soluble Guanylyl Cyclase ◽

Smooth Muscle Relaxation ◽

Membrane Cholesterol ◽

Caveolin 1

Erectile dysfunction is caused by a variety of pathogenic factors, particularly impaired formation and action of nitric oxide (NO). NO released from nerve endings and corpus cavernosum endothelial cells plays a crucial role in initiating and maintaining increased intracavernous pressure, penile vasodilatation, and penile erection. Classically, these effects are dependent on cGMP synthesized during activation of soluble guanylyl cyclase (sGC) by NO in smooth muscle cells. The enzyme NO synthase in endothelial cells has been localized to caveolae, small invaginations of the plasma membrane rich in cholesterol. Membrane cholesterol depletion impairs acetylcholine-induced relaxation in arteries attributed to an alteration in caveolar structure. It has been shown that sGC may be activated in endothelial caveolae contributing to vasodilation. We hypothesized that caveolae are the platform for sGC/cGMP signaling in cavernosum smooth muscle eliciting erection. Methyl-β-cyclodextrin, a pharmacological tool to deplete membrane cholesterol and disassemble caveolae, impaired rat erectile responses in vivo and cavernosum smooth muscle relaxation induced by the NO donor sodium nitroprusside and the sGC activator 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole in vitro. Methyl-β-cyclodextrin had no effect on cavernosum smooth muscle relaxation induced by NO released upon nerve stimulation or by exogenous cGMP. Furthermore, sGC and caveolin-1, the major coat protein of caveolae, were colocalized in rat corpus cavernosum sinusoidal endothelium. Electron microscopy indicated caveolae disruption in corpus cavernosum treated with methyl-β-cyclodextrin. In summary, our results provide evidence of compartmentalization of sGC in the caveolae of cavernosal endothelial cells contributing to NO signaling mediating smooth muscle relaxation and erection.

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Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation

Blood ◽

10.1182/blood-2011-03-341107 ◽

2011 ◽

Vol 118 (13) ◽

pp. 3670-3679 ◽

Cited By ~ 45

Author(s):

Guoying Zhang ◽

Binggang Xiang ◽

Anping Dong ◽

Radek C. Skoda ◽

Alan Daugherty ◽

...

Keyword(s):

Platelet Activation ◽

Guanylyl Cyclase ◽

Soluble Guanylyl Cyclase ◽

Whole Body ◽

No Donor ◽

No Donors ◽

Deficient Mice

AbstractNitric oxide (NO) stimulates cGMP synthesis by activating its intracellular receptor, soluble guanylyl cyclase (sGC). It is a currently prevailing concept that No and cGMP inhibits platelet function. However, the data supporting the inhibitory role of NO/sGC/cGMP in platelets have been obtained either in vitro or using whole body gene deletion that affects vessel wall function. Here we have generated mice with sGC gene deleted only in megakaryocytes and platelets. Using the megakaryocyte- and platelet-specific sGC-deficient mice, we identify a stimulatory role of sGC in platelet activation and in thrombosis in vivo. Deletion of sGC in platelets abolished cGMP production induced by either NO donors or platelet agonists, caused a marked defect in aggregation and attenuated secretion in response to low doses of collagen or thrombin. Importantly, megakaryocyte- and platelet-specific sGC deficient mice showed prolonged tail-bleeding times and impaired FeCl3-induced carotid artery thrombosis in vivo. Interestingly, the inhibitory effect of the NO donor SNP on platelet activation was sGC-dependent only at micromolar concentrations, but sGC-independent at millimolar concentrations. Together, our data demonstrate important roles of sGC in stimulating platelet activation and in vivo thrombosis and hemostasis, and sGC-dependent and -independent inhibition of platelets by NO donors.

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

Journal of Molecular Endocrinology ◽

10.1677/jme.1.02180 ◽

2006 ◽

Vol 37 (3) ◽

pp. 503-515 ◽

Cited By ~ 6

Author(s):

Yonghua Jiang ◽

Stanko S Stojilkovic

Keyword(s):

Guanylyl Cyclase ◽

Transcriptional Activity ◽

Soluble Guanylyl Cyclase ◽

Promoter Sequence ◽

Pituitary Cells ◽

Mobility Shift ◽

Binding Factor ◽

Rat Pituitary ◽

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 α1, α2, β1, and β2, is largely unknown. In this study, we identified the transcription start site of α1-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 α1-sGC subunit in resting pituitary cells.

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