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Cyclic guanosine monophosphate (cGMP) is a second messenger produced by the NO-sensitive guanylyl cyclase (NO-GC) enzyme. In platelets, the NO-GC/cGMP pathway inhibits aggregation. One aspect of the inhibitory mechanism involves changes in the cytoskeleton; however, the molecular mechanisms underlying platelet inhibition and its correlation with cytoskeletal cellular stiffness are poorly understood.We measured the cellular stiffness of individual platelets after treatment with the NO-GC stimulator riociguat or the NO-GC activator cinaciguat, using scanning ion conductance microscopy (SICM). We quantified changes in platelet shape using deep learning-based platelet morphometry. Cytoskeletal actin polymerization and platelet activation were measured by co-immunostaining F-actin and P-selectin, respectively. To test for clinical applicability of NO-GC stimulators in the context of increased thrombogenicity risk, we investigated the effect of riociguat on platelets from human immunodeficiency virus (HIV)-positive patients taking abacavir sulphate (ABC)-containing regimens, compared with HIV-negative volunteers.Stimulation of human and murine platelets with the NO-GC stimulator riociguat or with the NO-GC activator cinaciguat downregulated P-selectin expression, decreased F-actin polymerization, and decreased cellular stiffness by ≈50%, compared to vehicle control. In addition, platelets became more circular, indicating decreased activation. Riociguat did not cause any change in platelet aggregation or circularity in HIV-positive patients taking ABC-containing regimens.These results corroborate a functional role of the NO-GC enzyme in platelet biomechanics (cellular stiffness) in correlation with the inhibition of platelet activation and morphological changes. The observed changes in stiffness and platelet shape therefore demonstrate the possibility of pharmacologically targeting the NO-GC/cGMP pathway.

The mammalian genome encodes seven guanylyl cyclases, GC-A to GC-G, that are homodimeric transmembrane receptors activated by a diverse range of endogenous ligands. These enzymes convert guanosine-5'-triphosphate to the intracellular second messenger cyclic guanosine-3',5'-monophosphate (cyclic GMP). GC-A, GC-B and GC-C are expressed predominantly in the cardiovascular system, skeletal system and intestinal epithelium, respectively. GC-D and GC-G are found in the olfactory neuropepithelium and Grueneberg ganglion of rodents, respectively. GC-E and GC-F are expressed in retinal photoreceptors.

Increasing cGMP is a unique therapeutic principle, and drugs inhibiting cGMP-degrading enzymes or stimulating cGMP production are approved for the treatment of various diseases such as erectile dysfunction, coronary artery disease, pulmonary hypertension, chronic heart failure, irritable bowel syndrome, or achondroplasia. In addition, cGMP-increasing therapies are preclinically profiled or in clinical development for quite a broad set of additional indications, e.g., neurodegenerative diseases or different forms of dementias, bone formation disorders, underlining the pivotal role of cGMP signaling pathways. The fundamental understanding of the signaling mediated by nitric oxide-sensitive (soluble) guanylyl cyclase and membrane-associated receptor (particulate) guanylyl cyclase at the molecular and cellular levels, as well as in vivo, especially in disease models, is a key prerequisite to fully exploit treatment opportunities and potential risks that could be associated with an excessive increase in cGMP. Furthermore, human genetic data and the clinical effects of cGMP-increasing drugs allow back-translation into basic research to further learn about signaling and treatment opportunities. The biannual international cGMP conference, launched nearly 20 years ago, brings all these aspects together as an established and important forum for all topics from basic science to clinical research and pivotal clinical trials. This review summarizes the contributions to the “10th cGMP Conference on cGMP Generators, Effectors and Therapeutic Implications,” which was held in Augsburg in 2022 but will also provide an overview of recent key achievements and activities in the field of cGMP research.

The mammalian genome encodes seven guanylyl cyclases, GC-A to GC-G, that are homodimeric transmembrane receptors activated by a diverse range of endogenous ligands. These enzymes convert guanosine-5'-triphosphate to the intracellular second messenger cyclic guanosine-3',5'-monophosphate (cyclic GMP). GC-A, GC-B and GC-C are expressed predominantly in the cardiovascular system, skeletal system and intestinal epithelium, respectively. GC-D and GC-G are found in the olfactory neuropepithelium and Grueneberg ganglion of rodents, respectively. GC-E and GC-F are expressed in retinal photoreceptors.

Gastrointestinal dysfunctions in individuals with autism spectrum disorder are poorly understood, although they are common among this group of patients. FOXP1 haploinsufficiency is characterized by autistic behavior, language impairment, and intellectual disability, but feeding difficulties and gastrointestinal problems have also been reported. Whether these are primary impairments, the result of altered eating behavior, or side effects of psychotropic medication remains unclear. To address this question, we investigated Foxp1 +/− mice reflecting FOXP1 haploinsufficiency. These animals show decreased body weight and altered feeding behavior with reduced food and water intake. A pronounced muscular atrophy was detected in the esophagus and colon, caused by reduced muscle cell proliferation. Nitric oxide-induced relaxation of the lower esophageal sphincter was impaired and achalasia was confirmed in vivo by manometry. Foxp1 targets ( Nexn , Rbms3 , and Wls ) identified in the brain were dysregulated in the adult Foxp1 +/− esophagus. Total gastrointestinal transit was significantly prolonged due to impaired colonic contractility. Our results have uncovered a previously unknown dysfunction (achalasia and impaired gut motility) that explains the gastrointestinal disturbances in patients with FOXP1 syndrome, with potential wider relevance for autism.

3′,5′-cyclic guanosine monophosphate (cGMP) is a druggable second messenger regulating cell growth and survival in a plethora of cells and disease states, many of which are associated with hypoxia. For example, in myocardial infarction and heart failure (HF), clinical use of cGMP-elevating drugs improves disease outcomes. Although they protect mice from ischemia/reperfusion (I/R) injury, the exact mechanism how cardiac cGMP signaling is regulated in response to hypoxia is still largely unknown. By monitoring real-time cGMP dynamics in murine and human cardiomyocytes using in vitro and in vivo models of hypoxia/reoxygenation (H/R) and I/R injury combined with biochemical methods, we show that hypoxia causes rapid but partial degradation of cGMP-hydrolyzing phosphodiesterase-3A (PDE3A) protein via the autophagosomal-lysosomal pathway. While increasing cGMP in hypoxia prevents cell death, partially reduced PDE3A does not change the pro-apoptotic second messenger 3′,5′-cyclic adenosine monophosphate (cAMP). However, it leads to significantly enhanced protective effects of clinically relevant activators of nitric oxide-sensitive guanylyl cyclase (NO-GC). Collectively, our mouse and human data unravel a new mechanism by which cardiac cGMP improves hypoxia-associated disease conditions., Graphical abstract Image 1

AimThe role of platelets in atherosclerosis remains incompletely understood. Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here we sought to investigate the contribution of platelet sGC to atherosclerosis and the therapeutic potential of targeting sGC in atherosclerosis.Methods and ResultsWe genetically deleted sGC in platelets of atherosclerosis-prone Ldlr-/- mice. By intravital fluorescence microscopy such Pf4-Cre+Gucy1b1flox/floxLdlr-/- mice displayed enhanced leukocyte adhesion to atherosclerotic plaques in comparison with their litter mates. Moreover, histological and flow cytometry analyses revealed more numerous inflammatory leukocytes and larger plaque sizes in aortic tissue of Ldlr-/- mice lacking sGC in platelets. In vitro, supernatant from activated platelets lacking sGC promoted leukocyte adhesion to endothelial cells (EC) via enhanced EC activation. Using cytokine profiling, we identified reduced angiopoietin-1 release by Pf4-Cre+Gucy1b1flox/flox and human GUCY1A1 risk allele carrier platelets to be responsible for enhanced activation of EC and subsequent leukocyte adhesion. Pharmacological sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced recruitment of adoptively transferred leukocytes in Ldlr-/- mice fed a Western diet. Pharmacological sGC stimulation further reduced atherosclerotic plaque formation and vascular inflammation.ConclusionLoss of sGC in platelets contributes to atherosclerotic plaque formation via reduced release of the soluble factor angiopoietin-1 and, subsequently, enhanced leukocyte recruitment. Pharmacological sGC stimulation might represent a novel therapeutic strategy to prevent and treat CAD.Translational perspectiveReduced platelet soluble guanylyl cyclase activity contributes to atherosclerotic plaque formation and vascular inflammation. Stimulators of the soluble guanylyl cyclase, an emerging class of drugs already used in pulmonary hypertension and heart failure, are able to reduce atherosclerosis and inflammation in this preclinical model. Together with evidence from human genetics, our findings suggest a promising role of soluble guanylyl cyclase stimulation to prevent coronary artery disease.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15541. Catalytic receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

A large body of evidence indicates that nitric oxide (NO)/cGMP signaling essentially contributes to the processing of chronic pain. In general, NO-induced cGMP formation is catalyzed by 2 isoforms of guanylyl cyclase, NO-sensitive guanylyl cyclase 1 (NO-GC1) and 2 (NO-GC2). However, the specific functions of the 2 isoforms in pain processing remain elusive. Here, we investigated the distribution of NO-GC1 and NO-GC2 in the spinal cord and dorsal root ganglia, and we characterized the behavior of mice lacking either isoform in animal models of pain. Using immunohistochemistry and in situ hybridization, we demonstrate that both isoforms are localized to interneurons in the spinal dorsal horn with NO-GC1 being enriched in inhibitory interneurons. In dorsal root ganglia, the distribution of NO-GC1 and NO-GC2 is restricted to non-neuronal cells with NO-GC2 being the major isoform in satellite glial cells. Mice lacking NO-GC1 demonstrated reduced hypersensitivity in models of neuropathic pain, whereas their behavior in models of inflammatory pain was normal. By contrast, mice lacking NO-GC2 exhibited increased hypersensitivity in models of inflammatory pain, but their neuropathic pain behavior was unaltered. Cre-mediated deletion of NO-GC1 or NO-GC2 in spinal dorsal horn neurons recapitulated the behavioral phenotypes observed in the global knockout. Together, these results indicate that cGMP produced by NO-GC1 or NO-GC2 in spinal dorsal horn neurons exert distinct, and partly opposing, functions in chronic pain processing.

Mechanisms that limit thrombosis are poorly defined. One of the few known endogenous platelet inhibitors is nitric oxide (NO). NO activates NO sensitive guanylyl cyclase (NO-GC) in platelets, resulting in an increase of cyclic guanosine monophosphate (cGMP). Here we show, using cGMP sensor mice to study spatiotemporal dynamics of platelet cGMP, that NO-induced cGMP production in pre-activated platelets is strongly shear-dependent. We delineate a new mode of platelet-inhibitory mechanotransduction via shear-activated NO-GC followed by cGMP synthesis, activation of cGMP-dependent protein kinase I (cGKI), and suppression of Ca2+ signaling. Correlative profiling of cGMP dynamics and thrombus formation in vivo indicates that high cGMP concentrations in shear-exposed platelets at the thrombus periphery limit thrombosis, primarily through facilitation of thrombus dissolution. We propose that an increase in shear stress during thrombus growth activates the NO-cGMP-cGKI pathway, which acts as an auto-regulatory brake to prevent vessel occlusion, while preserving wound closure under low shear.

The mammalian genome encodes seven guanylyl cyclases, GC-A to GC-G, that are homodimeric transmembrane receptors activated by a diverse range of endogenous ligands. These enzymes convert guanosine-5'-triphosphate to the intracellular second messenger cyclic guanosine-3',5'-monophosphate (cyclic GMP). GC-A, GC-B and GC-C are expressed predominantly in the cardiovascular system, skeletal system and intestinal epithelium, respectively. GC-D and GC-G are found in the olfactory neuropepithelium and Grueneberg ganglion of rodents, respectively. GC-E and GC-F are expressed in retinal photoreceptors.

Cardiomyocyte β3-adrenoceptors (β3-ARs) coupled to soluble guanylyl cyclase (sGC)-dependent production of the second messenger 3’,5’-cyclic guanosine monophosphate (cGMP) have been shown to protect from heart failure. However, the exact localization of these receptors to fine membrane structures and subcellular compartmentation of β3-AR/cGMP signals underpinning this protection in health and disease remain elusive. Here, we used a Förster Resonance Energy Transfer (FRET)-based cGMP biosensor combined with scanning ion conductance microscopy (SICM) to show that functional β3-ARs are mostly confined to the T-tubules of healthy rat cardiomyocytes. Heart failure, induced via myocardial infarction, causes a decrease of the cGMP levels generated by these receptors and a change of subcellular cGMP compartmentation. Furthermore, attenuated cGMP signals led to impaired phosphodiesterase two dependent negative cGMP-to-cAMP cross-talk. In conclusion, topographic and functional reorganization of the β3-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.

Cyclic guanosine monophosphate (cGMP) is a unique second messenger molecule formed in different cell types and tissues. cGMP targets a variety of downstream effector molecules and, thus, elicits a very broad variety of cellular effects. Its production is triggered by stimulation of either soluble guanylyl cyclase (sGC) or particulate guanylyl cyclase (pGC); both enzymes exist in different isoforms. cGMP-induced effects are regulated by endogenous receptor ligands such as nitric oxide (NO) and natriuretic peptides (NPs). Depending on the distribution of sGC and pGC and the formation of ligands, this pathway regulates not only the cardiovascular system but also the kidney, lung, liver, and brain function; in addition, the cGMP pathway is involved in the pathogenesis of fibrosis, inflammation, or neurodegeneration and may also play a role in infectious diseases such as malaria. Moreover, new pharmacological approaches are being developed which target sGC- and pGC-dependent pathways for the treatment of various diseases. Therefore, it is of key interest to understand this pathway from scratch, beginning with the molecular basis of cGMP generation, the structure and function of both guanylyl cyclases and cGMP downstream targets; research efforts also focus on the subsequent signaling cascades, their potential crosstalk, and also the translational and, ultimately, the clinical implications of cGMP modulation. This review tries to summarize the contributions to the “9th International cGMP Conference on cGMP Generators, Effectors and Therapeutic Implications” held in Mainz in 2019. Presented data will be discussed and extended also in light of recent landmark findings and ongoing activities in the field of preclinical and clinical cGMP research.

Gastrointestinal dysfunctions in individuals with autism spectrum disorder are poorly understood, although they are common among this group of patients. FOXP1 haploinsufficiency is characterized by autistic behavior, language impairment, and intellectual disability, but feeding difficulties and gastrointestinal problems have also been reported. Whether these are primary impairments, the result of altered eating behavior, or side effects of psychotropic medication remains unclear. To address this question, we investigated Foxp1(+/−) mice reflecting FOXP1 haploinsufficiency. These animals show decreased body weight and altered feeding behavior with reduced food and water intake. A pronounced muscular atrophy was detected in the esophagus and colon, caused by reduced muscle cell proliferation. Nitric oxide-induced relaxation of the lower esophageal sphincter was impaired and achalasia was confirmed in vivo by manometry. Foxp1 targets (Nexn, Rbms3, and Wls) identified in the brain were dysregulated in the adult Foxp1(+/−) esophagus. Total gastrointestinal transit was significantly prolonged due to impaired colonic contractility. Our results have uncovered a previously unknown dysfunction (achalasia and impaired gut motility) that explains the gastrointestinal disturbances in patients with FOXP1 syndrome, with potential wider relevance for autism.

Cardiomyocyte β3-adrenoceptors (β3-ARs) coupled to soluble guanylyl cyclase (sGC)-dependent production of the second messenger 3’,5’-cyclic guanosine monophosphate (cGMP) have been shown to protect from heart failure. However, the exact localization of these receptors to fine membrane structures and subcellular compartmentation of β3-AR/cGMP signals underpinning this protection in health and disease remain elusive. Here, we used a Förster Resonance Energy Transfer (FRET)-based cGMP biosensor combined with scanning ion conductance microscopy (SICM) to show that functional β3-ARs are mostly confined to the T-tubules of healthy rat cardiomyocytes. Heart failure, induced via myocardial infarction, causes a decrease of the cGMP levels generated by these receptors and a change of subcellular cGMP compartmentation. Furthermore, attenuated cGMP signals led to impaired phosphodiesterase two dependent negative cGMP-to-cAMP cross-talk. In conclusion, topographic and functional reorganization of the β3-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.

The nitric oxide (NO)–sensitive soluble guanylyl cyclase (sGC) is a heterodimeric enzyme with an α and β subunit. NO binds to heme of the β1-subunit of sGC, activates the enzyme in the reduced heme iron state in vascular smooth muscle cells (VSMCs), and generates cGMP-inducing vasodilatation and suppression of VSMC proliferation. In the complex tumor milieu with higher levels of reactive oxygen species (ROS), sGC heme iron may become oxidized and insensitive to NO. To change sGC from an NO-insensitive to NO-sensitive state or NO-independent manner, protein expression of sGC in VSMC is required. Whether sGCα1β1 exists at the protein level in arterial VSMCs of oropharyngeal squamous cell carcinoma (OPSCC) is unknown. In addition, whether differences in the genetic profile between human papillomavirus (HPV)–positive and HPV-negative OPSCC contributes to the regulation of sGCα1β1 is unclear. Therefore, we compared the effects of HPV-positive and HPV-negative OPSCC on the expression of sGCα1β1 in arterial VSMCs from tumor-free and tumor-containing regions of human tissue sections using quantitative immunohistochemistry. In comparison to the tumor-free region, we found a decrease in expression of both α1- and β1-subunits in the arterial VSMC layer of the tumor-containing areas. The OPSCC-induced significant downregulation of the α1- and β1-subunits of sGC in arterial VSMC was HPV-independent. We conclude that the response of sGC to NO in tumor arterial VSMCs may be impaired by oxidation of the heme of the β1-subunit, and thus, α1- and β1-subunits of sGC could be targeted to degradation under oxidative stress in OPSCC in an HPV-independent manner. The degradation of sGCα1β1 in VSMCs may result in increased proliferation of VSMCs, promoting tumor arteriogenesis in OPSCC. This can be interrupted by preserving the active heterodimer sGCα1β1 in arterial VSMCs.

Phosphodiesterase 3 (PDE3) exists in two isoforms (PDE3A and PDE3B) and is known to act as cGMP-inhibited cAMP-degrading PDE. Therefore, PDE3 may likely be involved in the interaction between the two second messenger pathways. NO-sensitive guanylyl cyclase (NO-GC) is the most important cytosolic generator of cGMP. Here, we investigated the effect of NO-GC deletion on PDE3A-mediated signaling in animals lacking NO-GC either globally (GCKO) or specifically in smooth muscle cells (SMC-GCKO). PDE3A expression is detected in murine aortic smooth muscle, platelets, and heart tissue. Expression and activity of PDE3A in aortae from GCKO and SMC-GCKO mice was reduced by approx. 50% compared to that in control animals. PDE3A downregulation can be linked to the reduction in NO-GC and is not an effect of the increased blood pressure levels resulting from NO-GC deletion. Despite the different PDE3A expression levels, smooth muscle relaxation induced by forskolin to stimulate cAMP signaling was similar in all genotypes. Basal and forskolin-stimulated cAMP levels in aortic tissue were not different between KO and control strains. However, the potency of milrinone, a selective inhibitor of PDE3A, to induce relaxation was higher in aortae from GCKO and SMC-GCKO than that in aorta from control animals. These data were corroborated by the effect of milrinone in vivo, which led to an increase in systolic blood pressure in both KO strains but not in control mice. We conclude that NO-GC modulates PDE3A expression and activity in SMC in vivo conceivably to preserve functional cAMP signaling.

Although the Nobel Prize for the discovery of nitric oxide (NO) dates back almost 20 years now, the knowledge about cGMP signaling is still constantly increasing. It looks even so that our understanding of the role of the soluble guanylyl cyclase (sGC) and particulate guanylyl cyclase (pGC) in health and disease is in many aspects at the beginning and far from being understood. This holds even true for the therapeutic impact of innovative drugs acting on both the NO/sGC and the pGC pathways. Since cGMP, as second messenger, is involved in the pathogenesis of numerous diseases within the cardiovascular, pulmonary, renal, and endocrine systems and also plays a role in neuronal, sensory, and tumor processes, drug applications might be quite broad. On the 8th International Conference on cGMP, held in Bamberg, Germany, world leading experts came together to discuss these topics. All aspects of cGMP research from the basic understanding of cGMP signaling to clinical applicability were discussed in depth. In addition, present and future therapeutic applications of cGMP-modulating pharmacotherapy were presented (http://www.cyclicgmp.net/index.html).

Nitric oxide (NO) activates the NO-sensitive soluble guanylate cyclase (NO-GC, sGC) and triggers intracellular signaling pathways involving cGMP. For survival of cochlear hair cells and preservation of hearing, NO-mediated cascades have both protective and detrimental potential. Here we examine the cochlear function of mice lacking one of the two NO-sensitive guanylate cyclase isoforms [NO-GC1 knockout (KO) or NO-GC2 KO]. The deletion of NO-GC1 or NO-GC2 did not influence electromechanical outer hair cell (OHC) properties, as measured by distortion product otoacoustic emissions, neither before nor after noise exposure, nor were click- or noise-burst-evoked auditory brainstem response thresholds different from controls. Yet inner hair cell (IHC) ribbons and auditory nerve responses showed significantly less deterioration in NO-GC1 KO and NO-GC2 KO mice after noise exposure. Consistent with a selective role of NO-GC in IHCs, NO-GC β1 mRNA was found in isolated IHCs but not in OHCs. Using transgenic mice expressing the fluorescence resonance energy transfer-based cGMP biosensor cGi500, NO-induced elevation of cGMP was detected in real-time in IHCs but not in OHCs. Pharmacologic long-term treatment with a NO-GC stimulator altered auditory nerve responses but did not affect OHC function and hearing thresholds. Interestingly, NO-GC stimulation exacerbated the loss of auditory nerve response in aged animals but attenuated the loss in younger animals. We propose NO-GC2 and, to some degree, NO-GC1 as targets for early pharmacologic prevention of auditory fiber loss (synaptopathy). Both isoforms provide selective benefits for hearing function by maintaining the functional integrity of auditory nerve fibers in early life rather than at old age.

The mammalian genome encodes transmembrane and soluble receptor guanylyl cyclases, both of which have enzyme activities which convert guanosine-5'-triphosphate to the intracellular second messenger cyclic guanosine-3',5'-monophosphate (cyclic GMP).

Background Nitric oxide (NO) mediates inhibitory neurotransmission and is a critical component of neuronal programs that generate propulsive contractions. NO acts via its receptor NO-sensitive guanylyl cyclase (NO-GC) which is expressed in smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Organ bath studies with colonic rings from NO-GC knockout mice (GCKO) have indicated NO-GC to modulate spontaneous contractions. The cell-specific effects of NO-GC on the dominant pan-colonic propulsive contraction, the long distance contractions (LDCs), of whole colon preparations have not yet been described. Methods Contractions of whole colon preparations from wild type (WT), global, and cell-specific GCKO were recorded. After transformation into spatiotemporal maps, motility patterns were analyzed. Simultaneous perfusion of the colon enabled the correlation of outflow with LDCs to analyze contraction efficiency. Key results Deletion of NO-GC in both ICC and SMC (ie, in GCKO and SMC/ICC-GCKO) caused loss of typical LDC activity and instead generated high-frequency LDC-like contractions with inefficient propulsive activity. Frequency was also increased in WT, SMC-GCKO, and ICC-GCKO colon in the presence of L-NAME to block neuronal NO synthase. LDC efficiency was dependent on NO-GC in SMC as it was reduced in GCKO, SMC-GCKO, and ICC/SMC-GCKO colon; LDC efficiency was decreased in all genotypes in the presence of L-NAME. Conclusions and inferences NO/cGMP signaling is critical for normal peristaltic movements; as NO-GC in both SMC and ICC is essential, both cell types appear to work in synchrony. The efficiency of contractions to expel fluid is particularly influenced by NO-GC in SMC.

Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the &alpha, 1- and &beta, 1-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the &alpha, 1-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both &alpha, 1-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption.

In the gastrointestinal (GI) tract, nitric oxide (NO) has been shown over the last 25 years to exert a prominent function as inhibitory neurotransmitter. Apart from the regulation of secretion and resorption, NO from nitrergic neurons has been demonstrated to be crucial for GI smooth muscle relaxation and motility. In fact, several human diseases such as achalasia, gastroparesis, slow transit constipation or Hirschsprung's disease may involve dysfunctional nitrergic signaling. Most of NO's effects as neurotransmitter are mediated by NO-sensitive guanylyl cyclase (NO-GC) and further transduced by cGMP-dependent mechanisms. In contrast to the vascular system where NO from the endothelium induces relaxation by acting on NO-GC solely in smooth muscle cells, GI tissues contain several different NO-GCexpressing cell types that include smooth muscle cells, interstitial cells of Cajal and fibroblast-like cells. Based on this diverse localization of the NO receptor, the exact pathway(s) leading to NO-induced relaxation are still unknown. Global and cell-specific knockout mouse strains have been generated that lack enzymes participating in nitrergic signaling. These animals have been helpful in examining the role of NO in smooth muscle of the GI tract. Here, we discuss the current knowledge on NO-mediated mechanisms in the relaxation of GI smooth muscle in stomach, small and large intestine including sphincters. Special focus is placed on the integration of nitrergic signals by specialized cell types within the gut smooth muscle layers.

During the past decade, our knowledge on the physiology, pathophysiology, basic pharmacology, and clinical pharmacology of the second messenger (cGMP) has increased tremendously. It is now well-established that cGMP, generated by soluble and particulate guanylate cyclases, is highly compartmentalized in cells and regulates numerous body functions. New cGMP-regulated physiological functions include meiosis and temperature perception. cGMP is involved in the genesis of numerous pathologies including cardiovascular, pulmonary, endocrine, metabolic, neuropsychiatric, eye, and tumor diseases. Several new clinical uses of stimulators and activators of soluble guanylate cyclase and of phosphodiesterase inhibitors such as heart failure, kidney failure, cognitive disorders, obesity bronchial asthma, and osteoporosis are emerging. The combination of neprilysin inhibitors-enhancing stimulation of the particulate guanylate cyclase pathway by preventing natriuretic peptide degradation-with angiotensin AT1 receptor antagonists constitutes a novel promising strategy for heart failure treatment. The role of oxidative stress in cGMP signaling, application of cGMP sensors, and gene therapy for degenerative eye diseases are emerging topics. It is anticipated that cGMP research will further prosper over the next years and reach out into more and more basic and clinical disciplines.

Background and Purpose Stimulation of soluble guanylyl cyclase (sGC) is a valuable therapeutic strategy for the treatment of several cardiovascular diseases. The sGC stimulator riociguat has been approved for the treatment of two forms of pulmonary hypertension. Platelets contain large amounts of sGC and play a key role in the regulation of haemostasis. Therefore, we investigated the effects of riociguat on platelet function. Experimental Approach The effect of riociguat treatment on human platelet activation and aggregation was investigated. The sGC-specific effects of riociguat were determined by comparing wild-type and platelet-specific sGC-knockout mice. Key Results Riociguat induced cGMP synthesis and subsequent PKG activation in human platelets, suggesting that the inhibitory effects are mediated by cGMP signalling. This finding was confirmed when sGC-knockout platelets were not inhibited by riociguat. In washed human platelets, 100 nM riociguat reduced ADP-induced GPIIb/IIIa activation, while a 10-fold higher concentration was required to reduce convulxin-stimulated GPIIb/IIIa activation. Riociguat inhibited ADP-induced platelet shape change and aggregation, while ATP-induced shape change remained unaffected. However, in PRP and whole blood, 50–100 μM riociguat was required to inhibit platelet activation and aggregation. Riociguat in combination with iloprost significantly inhibited platelet aggregation, even in whole blood. Conclusions and Implications Riociguat inhibits platelet activation in whole blood only at concentrations above 50 μM, while the plasma concentrations in riociguat-treated patients are 150 to 500 nM. This finding indicates that riociguat treatment does not affect platelet function in patients. Nevertheless, the possibility that riociguat acts synergistically with iloprost to inhibit platelet activation should be considered.

Introduction: The genomic locus harboring the GUCY1A3 gene has been associated with coronary artery disease (CAD) with the risk variant reducing GUCY1A3 expression via allele-specific transcription factor binding. The gene encodes the alpha 1 -subunit of the soluble guanylyl cyclase (sGC) which produces the second messenger cyclic guanosine monophosphate (cGMP) upon activation by nitric oxide, thereby influencing the function of vascular smooth muscle cells and platelets. Aim: To investigate whether a reduction in sGC expression specifically in platelets influences atherosclerotic plaque formation. Methods: By crossbreeding Pf4-Cre and sGC β 1 fl/fl mice, platelet-specific sGC knockout (PS-sGC -/- ) mice were generated. These were crossbred with Ldlr -/- mice to generate PS-sGC -/- Ldlr -/- mice. PS-sGC -/- Ldlr -/- and Ldlr -/- mice were fed a high-cholesterol diet (HCD) for 10 weeks. Serial sections of the aortic root were analyzed by Masson-Trichrome staining. Inflammatory cells in aortae were assessed using flow cytometry. In vitro adhesion assays using wildtype endothelial cells (EC) and monocytes were performed in presence of plasma of activated platelets. Results: PS-sGC -/- Ldlr -/- (n=13) mice displayed enhanced total plaque size compared to Ldlr -/- (n=15) mice (247·10 3 vs. 190·10 3 μm 2 , phigh monocytes (366 vs. 207, p-/- Ldlr -/- compared to Ldlr -/- mice indicating enhanced recruitment of these cells in PS-sGC -/- Ldlr -/- mice. In in vitro adhesion assays, incubation of mouse aortic EC and monocytes with plasma from PS-sGC -/- platelets resulted in enhanced adhesion of monocytes to EC compared to plasma from wildtype platelets (13%, p Conclusion and Outlook: Platelet-specific knockout of sGC led to increased atherosclerotic plaque formation and inflammation. In vitro adhesion assay results point to an enhanced stimulation of leukocyte adhesion to endothelial cells in PS-sGC -/- mice. Hence, factors released by platelets secondary to reduced sGC expression could mediate increased risk of CAD. Inhibiting platelet activation in carriers of the human risk variant might therefore be a promising therapeutic strategy to reduce risk of CAD.

ObjectivesStimulators of soluble guanylate cyclase (sGC) are currently investigated in clinical trials for the treatment of fibrosis in systemic sclerosis (SSc). In this study, we aim to investigate the role of protein kinases G (PKG) as downstream mediators of sGC–cyclic guanosine monophosphate (cGMP) in SSc.MethodsMice with combined knockout of PKG1 and 2 were challenged with bleomycin and treated with the sGC stimulator BAY 41-2272. Fibroblasts were treated with BAY 41-2272 and with the PKG inhibitor KT 5823.ResultsPKG1 and 2 are upregulated in SSc in a transforming growth factor-β1 (TGFβ1)-dependent manner, as an attempt to compensate for the decreased signalling through the sGC–cGMP–PKG pathway. Inhibition or knockout of PKG1 and 2 abrogates the inhibitory effects of sGC stimulation on fibroblast activation in a SMAD-independent, but extracellular signal-regulated kinase (ERK)-dependent manner. In vivo, sGC stimulation fails to prevent bleomycin-induced fibrosis in PKG1 and 2 knockout mice.ConclusionsOur data provide evidence that PKGs are essential mediators of the antifibrotic effects of sGC stimulators through interfering with non-canonical TGFβ signalling. TGFβ1 promotes its profibrotic effects through inhibition of sGC–cGMP–PKG signalling, sGC stimulation exerts its antifibrotic effects by inhibition of TGFβ1-induced ERK phosphorylation.

In the enteric nervous systems, NO is released from nitrergic neurons as a major inhibitory neurotransmitter. NO acts via NO-sensitive guanylyl cyclase (NO-GC), which is found in different gastrointestinal (GI) cell types including smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC). The precise mechanism of nitrergic signalling through these two cell types to regulate colonic spontaneous contractions is not fully understood yet. In the present study we investigated the impact of endogenous and exogenous NO on colonic contractile motor activity using mice lacking nitric oxide-sensitive guanylyl cyclase (NO-GC) globally and specifically in SMCs and ICC. Longitudinal smooth muscle of proximal colon from wild-type (WT) and knockout (KO) mouse strains exhibited spontaneous contractile activity ex vivo. WT and smooth muscle-specific guanylyl cyclase knockout (SMC-GCKO) colon showed an arrhythmic contractile activity with varying amplitudes and frequencies. In contrast, colon from global and ICC-specific guanylyl cyclase knockout (ICC-GCKO) animals showed a regular contractile rhythm with constant duration and amplitude of the rhythmic contractions. Nerve blockade (tetrodotoxin) or specific blockade of NO signalling (L-NAME, ODQ) did not significantly affect contractions of GCKO and ICC-GCKO colon whereas the arrhythmic contractile patterns of WT and SMC-GCKO colon were transformed into uniform motor patterns. In contrast, the response to electric field-stimulated neuronal NO release was similar in SMC-GCKO and global GCKO. In conclusion, our results indicate that basal enteric NO release acts via myenteric ICC to influence the generation of spontaneous contractions whereas the effects of elevated endogenous NO are mediated by SMCs in the murine proximal colon.

Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO·), confers unique physiological effects including vasorelaxation and enhanced cardiac contractility. These features have spawned current pharmaceutical development of HNO donors as heart failure therapeutics. HNO interacts with selective redox sensitive cysteines to effect signaling but is also proposed to activate soluble guanylate cyclase (sGC) in vitro to induce vasodilation and potentially enhance contractility. Here, we tested whether sGC stimulation is required for these HNO effects in vivo and if HNO also modifies a redox-sensitive cysteine (C42) in protein kinase G-1α to control vasorelaxation. Intact mice and isolated arteries lacking the sGC-β subunit (sGCKO, results in full sGC deficiency) or expressing solely a redox-dead C42S mutant protein kinase G-1α were exposed to the pure HNO donor, CXL-1020. CXL-1020 induced dose-dependent systemic vasodilation while increasing contractility in controls; however, vasodilator effects were absent in sGCKO mice whereas contractility response remained. The CXL-1020 dose reversing 50% of preconstricted force in aortic rings was ≈400-fold greater in sGCKO than controls. Cyclic-GMP and cAMP levels were unaltered in myocardium exposed to CXL-1020, despite its inotropic-vasodilator activity. In protein kinase G-1α C42S mice, CXL-1020 induced identical vasorelaxation in vivo and in isolated aortic and mesenteric vessels as in littermate controls. In both groups, dilation was near fully blocked by pharmacologically inhibiting sGC. Thus, sGC and cGMP-dependent signaling are necessary and sufficient for HNO-induced vasodilation in vivo but are not required for positive inotropic action. Redox modulation of protein kinase G-1α is not a mechanism for HNO-mediated vasodilation.

Oesophageal achalasia is a disease known to result from reduced relaxation of the lower oesophageal sphincter (LES). Nitric oxide (NO) is one of the main inhibitory transmitters. NO-sensitive guanylyl cyclase (NO-GC) acts as the key target of NO and, by the generation of cGMP, mediates nitrergic relaxation in the LES. To date, the exact mechanism of nitrergic LES relaxation is still insufficiently elucidated. To clarify the role of NO-GC in LES relaxation, we used cell-specific knockout (KO) mouse lines for NO-GC. These include mice lacking NO-GC in smooth muscle cells (SMC-GCKO), in interstitial cells of Cajal (ICC-GCKO) and in both SMC/ICC (SMC/ICC-GCKO). We applied oesophageal manometry to study the functionality of LES in vivo. Isometric force studies were performed to monitor LES responsiveness to exogenous NO and electric field stimulation of intrinsic nerves in vitro. Cell-specific expression/deletion of NO-GC was monitored by immunohistochemistry. Swallowing-induced LES relaxation is strongly reduced by deletion of NO-GC in ICC. Basal LES tone is affected by NO-GC deletion in either SMC or ICC. Lack of NO-GC in both cells leads to a complete interruption of NO-induced relaxation and, therefore, to an achalasia-like phenotype similar to that seen in global GCKO mice. Our data indicate that regulation of basal LES tone is based on a dual mechanism mediated by NO-GC in SMC and ICC whereas swallow-induced LES relaxation is mainly regulated by nitrergic mechanisms in ICC.

AIMS: It has been suggested that the nitric oxide-sensitive guanylyl cyclase (NO-GC)/cyclic guanosine monophosphate (cGMP)-dependent signalling pathway affords protection against cardiac damage during acute myocardial infarction (AMI). It is, however, not clear whether the NO-GC/cGMP system confers its favourable effects through a mechanism located in cardiomyocytes (CMs). The aim of this study was to evaluate the infarct-limiting effects of the endogenous NO-GC in CMs in vivo. METHODS AND RESULTS: Ischemia/reperfusion (I/R) injury was evaluated in mice with a CM-specific deletion of NO-GC (CM NO-GC KO) and in control siblings (CM NO-GC CTR) subjected to an in vivo model of AMI. Lack of CM NO-GC resulted in a mild increase in blood pressure but did not affect basal infarct sizes after I/R. Ischemic postconditioning (iPost), administration of the phosphodiesterase-5 inhibitors sildenafil and tadalafil as well as the NO-GC activator cinaciguat significantly reduced the amount of infarction in control mice but not in CM NO-GC KO littermates. Interestingly, NS11021, an opener of the large-conductance and Ca2+-activated potassium channel (BK), an important downstream effector of cGMP/cGKI in the cardiovascular system, protects I/R-exposed hearts of CM NO-GC proficient and deficient mice. CONCLUSIONS: These findings demonstrate an important role of CM NO-GC for the cardioprotective signalling following AMI in vivo. CM NO-GC function is essential for the beneficial effects on infarct size elicited by iPost and pharmacological elevation of cGMP; however, lack of CM NO-GC does not seem to disrupt the cardioprotection mediated by the BK opener NS11021.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) in young adults that has serious negative socioeconomic effects. In addition to symptoms caused by CNS pathology, the majority of MS patients frequently exhibit gastrointestinal dysfunction, which was previously either explained by the presence of spinal cord lesions or not directly linked to the autoimmune etiology of the disease. Here, we studied the enteric nervous system (ENS) in a B cell- and antibody-dependent mouse model of MS by immunohistochemistry and electron microscopy at different stages of the disease. ENS degeneration was evident prior to the development of CNS lesions and the onset of neurological deficits in mice. The pathology was antibody mediated and caused a significant decrease in gastrointestinal motility, which was associated with ENS gliosis and neuronal loss. We identified autoantibodies against four potential target antigens derived from enteric glia and/or neurons by immunoprecipitation and mass spectrometry. Antibodies against three of the target antigens were also present in the plasma of MS patients as confirmed by ELISA. The analysis of human colon resectates provided evidence of gliosis and ENS degeneration in MS patients compared to non-MS controls. For the first time, this study establishes a pathomechanistic link between the well-established autoimmune attack on the CNS and ENS pathology in MS, which might provide a paradigm shift in our current understanding of the immunopathogenesis of the disease with broad diagnostic and therapeutic implications.

Nitric oxide (NO) is a major inhibitory neurotransmitter in the gastrointestinal (GI) tract. Its main effector, NO-sensitive guanylyl cyclase (NO-GC), is expressed in several GI cell types, including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), and fibroblast-like cells. Up to date, the interplay between neurons and these cells to initiate a nitrergic inhibitory junction potential (IJP) is unclear. Here, we investigate the origin of the nitrergic IJP in murine fundus and colon. IJPs were determined in fundus and colon SMC of mice lacking NO-GC globally (GCKO) and specifically in SMC (SM-GCKO), ICC (ICC-GCKO), and both SMC/ICC (SM/ICC-GCKO). Nitrergic IJP was abolished in ICC-GCKO fundus and reduced in SM-GCKO fundus. In the colon, the amplitude of nitrergic IJP was reduced in ICC-GCKO, whereas nitrergic IJP in SM-GCKO was reduced in duration. These results were corroborated by loss of the nitrergic IJP in global GCKO. In conclusion, our results prove the obligatory role of NO-GC in ICC for the initiation of an IJP. NO-GC in SMC appears to enhance the nitrergic IJP, resulting in a stronger and prolonged hyperpolarization in fundus and colon SMC, respectively. Thus NO-GC in both cell types is mandatory to induce a full nitrergic IJP. Our data from the colon clearly reveal the nitrergic IJP to be biphasic, resulting from individual inputs of ICC and SMC.

The action of nitric oxide (NO) to stimulate NO-sensitive guanylyl cyclase (NO-GC), followed by production of cGMP, and eventually to cause smooth muscle relaxation is well known. In the lower urinary tract (LUT), in contrast to the cardiovascular system and the gastrointestinal tract, specific localization in combination with function of NO-GC has not been investigated to date. Consequently, little is known about the mechanisms regulating relaxation of the lower urinary tract in general and the role of NO-GC-expressing cells in particular. To study the distribution and function of NO-GC in the murine lower urinary tract, we used internal urethral sphincter and bladder detrusor from global (GCKO) and smooth muscle cell-specific (SM-GCKO) NO-GC knock-out mice for immunohistochemical analyses and organ bath experiments. In urethral sphincter, NO-GC-positive immunofluorescence was confined to smooth muscle cells (SMCs). Deletion of NO-GC in SMCs abolished NO-induced relaxation. In bladder detrusor, exposure to NO did not cause relaxation although immunohistochemistry uncovered the existence of NO-GC in the tissue. In contrast to the urethral sphincter, expression of NO-GC in bladder detrusor was limited to platelet-derived growth factor receptor α (PDGFRα)-positive interstitial cells. In conclusion, NO-GC found in SMCs of the urethral sphincter mediates NO-induced relaxation; bladder detrusor is unique as NO-GC is not expressed in SMCs and, thus, NO does not induce relaxation. Nevertheless, NO-GC expression was found in PDGFRα-positive interstitial cells of the murine bladder with an as yet unknown function. Further investigation is needed to clarify the role of NO-GC in the detrusor.