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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

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

Background and Purpose Nitric oxide (NO) is known to activate NO-sensitive guanylyl cyclase (NO-GC) and to elicit cGMP production. However, NO has also been proposed to induce cGMP-independent effects. It is accepted practice to use specific NO-GC inhibitors, such as ODQ or NS2028, to assess cGMP-dependent NO effects. Consequently, NO-induced reactions seen in the presence of these inhibitors commonly serve as an affirmation of cGMP independence. Experimental Approach We evaluated the use of ODQ to discriminate between cGMP-dependent and cGMP-independent NO effects. NO-GC-expressing HEK cells, platelets and tissues from wild type (WT) and NO-GC-deficient mice (GCKO) were used. Key Results NO donors led to accumulation of cGMP in platelets and GC-expressing HEK cells and induced phosphorylation of the vasodilator-stimulated phosphoprotein in platelets; both effects were reduced by ODQ. High concentrations of NO donors, however, overrode the inhibitory effect of ODQ. Correspondingly, ODQ inhibited but did not fully eliminate NO-induced relaxation of aorta and fundus from WT mice. Relaxation induced by endogenously released NO was fully or partially inhibited by ODQ in fundus and aorta, respectively. In aorta and fundus of GCKO mice NO-induced relaxation was absent and served as standard for complete NO-GC inhibition. Conclusions and Implications High NO concentrations can overcome the inhibitory effect of ODQ on NO-GC. Smooth muscle relaxation induced by NO donors/endogenously released NO in the presence of ODQ in WT was absent in GCKO animals indicating involvement of NO-GC. Accordingly, NO-induced effects in the presence of ODQ do not necessarily prove cGMP independence.

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

Key points • Erectile dysfunction may result from reduced or non-functional nitric oxide (NO)/cGMP-mediated signalling. Mice lacking NO synthases are fertile whereas mice deficient in cGMP-dependent protein kinase I suffer from erectile dysfunction. • To clarify this discrepancy we performed studies on the corpus cavernosum of male mice lacking the NO receptor NO-sensitive guanylyl cyclase (NO-GC) either globally or specifically in smooth muscle cells. • NO released from NO donors as well as from nitrergic neurons failed to relax precontracted corpus cavernosum from mice lacking NO-GC either globally or specifically in smooth muscle; to our surprise, males from both knockout lines were fertile. • Our data show that deletion of the NO receptor specifically in smooth muscle cells abolishes NO-induced corpus cavernosum relaxation but does not lead to infertility. Abstract Nitric oxide (NO) and cGMP have been shown to be important mediators of penile erection. Erectile dysfunction may result from reduced or non-functional signal transduction within this cascade. There is, however, some inconsistency in the available data as mice lacking NO synthases (endothelial and neuronal nitric oxide synthase, or both) appear to be fertile whereas mice deficient in cGMP-dependent protein kinase I (PKGI) suffer from erectile dysfunction. To clarify this discrepancy we performed studies on mice lacking the NO receptor NO-sensitive guanylyl cyclase (NO-GC). In addition, we generated cell-specific NO-GC knockout (KO) lines to investigate the function of NO in individual cell types. NO-GC was specifically deleted in smooth muscle or endothelial cells (SM-guanylyl cyclase knockout (SM-GCKO) and EC-GCKO, respectively) and these KO lines were compared with total knockouts (GCKO) and wild-type animals. We investigated expression of NO-GC, NO-induced relaxation of corpus cavernosum smooth muscle and their ability to generate offspring. NO-GC-positive immunostaining was detected in smooth muscle and endothelial cells of murine corpus cavernosum but not in interstitial cells of Cajal. NO released from NO donors as well as from nitrergic neurons failed to relax precontracted corpus cavernosum from GCKO mice in organ bath experiments. Similar results were obtained in corpus cavernosum from SM-GCKO mice whereas deletion of NO-GC in endothelial cells did not affect relaxation. The lack of NO-induced relaxation in GCKO animals was not compensated for by guanosine 3′,5′-cyclic monophosphate (cGMP) signalling. To our surprise, GCKO males were fertile although their ability to produce offspring was decreased. Our data show that deletion of NO-GC specifically in smooth muscle cells abolishes NO-induced corpus cavernosum relaxation but does not lead to infertility.

Stimulators of the soluble guanylyl cyclase (sGC) are emerging therapeutic agents in cardiovascular diseases. Genetic alterations of the GUCY1A3 gene, which encodes the alpha(1) subunit of the sGC, are associated with coronary artery disease. Studies investigating sGC stimulators in subjects with CAD and carrying risk-related variants in sGC are, however, lacking. Here, we functionally investigate the impact of coding GUCY1A3 variants on sGC activity and the therapeutic potential of sGC stimulators in vitro. In addition to a known loss-of-function variant, eight coding variants in GUCY1A3 were cloned and expressed in HEK 293 cells. Protein levels and dimerization capability with the beta(1) subunit were analysed by immunoblotting and co-immunoprecipitation, respectively. All alpha(1) variants found in MI patients dimerized with the beta(1) subunit. Protein levels were reduced by 72 % in one variant (p < 0.01). Enzymatic activity was analysed using cGMP radioimmunoassay after stimulation with a nitric oxide (NO) donor. Five variants displayed decreased cGMP production upon NO stimulation (p < 0.001). The addition of the sGC stimulator BAY 41-2272 increased cGMP formation in all of these variants (p < 0.01). Except for the variant leading to decreased protein level, cGMP amounts reached the wildtype NO-induced level after addition of BAY 41-2272. In conclusion, rare coding variants in GUCY1A3 lead to reduced cGMP formation which can be rescued by a sGC stimulator in vitro. These results might therefore represent the starting point for discovery of novel treatment strategies for patients at risk with coding GUCY1A3 variants.

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

SummaryPlatelet activation is an irreversible process resulting in platelet apoptosis and necrosis, and circulating platelets contain many components of the apoptotic machinery. Cyclic guanosine monophosphate (cGMP) generated by nitric oxide (NO) activated soluble guanylyl cyclase (sGC) plays a crucial role in preventing platelet activation. However, in addition to activation of sGC, cGMP-independent NO effects in platelets have been described. To differentiate between cGMP-dependent and -independent NO effects on platelet apoptosis and reactive oxygen species (ROS) production, we generated platelet-specific sGC-deficient mice (PS-GCKO). Platelet apoptosis was induced by a combination of thrombin/convulxin (Thr/Cvx) and assessed by phosphatidylserine (PS) surface exposure, and loss of the mitochondrial membrane potential. NO-induced inhibition of PS externalisation was mediated only by cGMP-dependent mechanisms. Inhibition of the mitochondrial membrane potential decrease at low NO concentration was also cGMP-dependent but became cGMP-independent at high NO concentrations. In contrast, inhibition of ROS formation at any NO concentration was mediated by cGMP-independent mechanisms, very likely due to direct radical scavenging. NO inhibits platelet apoptosis by cGMP-dependent mechanisms and ROS production by cGMP-independent mechanisms. The PS-GCKO mouse model is an important tool for the differentiation of cGMP-dependent and -independent NO effects on platelets.

See also Gordge MP. Nitric oxide: a one-trick pony? This issue, pp 1340–2. Summary. Background: The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling cascade is involved in the precise regulation of platelet responses. NO released from the endothelium is known to activate NO-sensitive guanylyl cyclase (NO-GC) in platelets. By the generation of cGMP and subsequent activation of cGMP-dependent protein kinase (PKG), NO-GC mediates the reduction of the intracellular calcium and inhibits platelet adhesion and aggregation. However, NO has been postulated to influence these platelet functions also via cGMP-independent mechanisms. Objective: We studied the effect of NO on platelets lacking NO-sensitive guanylyl cyclase with regards to aggregation, adhesion, calcium mobilization and bleeding time. Methods and results: Here, we show that NO signaling leading to inhibition of agonist-induced platelet aggregation is totally abrogated in platelets from mice deficient in NO-GC (GCKO). Even at millimolar concentrations none of the several different NO donors inhibited collagen-induced aggregation of GCKO platelets. In addition, NO neither affected adenosine 5′-diphosphate (ADP)-induced adhesion nor thrombin-induced calcium release in GCKO platelets. Although the NO-induced cGMP signal transduction was totally abrogated cyclic adenosine monophosphate (cAMP) signaling was still functional; however, cGMP/cAMP crosstalk was disturbed on the level of phosphodiesterase type 3 (PDE3). These in vitro data are completed by a reduced bleeding time indicating the lack of NO effect in vivo. Conclusions: We conclude that NO-GC is the only NO receptor in murine platelets mediating the inhibition of calcium release, adhesion and aggregation: lack of the enzyme leads to disturbance of primary hemostasis.

Background Regulation of gastrointestinal motility involves excitatory and inhibitory neurotransmission. Nitric oxide (NO), the major inhibitory neurotransmitter, acts via its receptor NO-sensitive guanylyl cyclase (NO-GC). In the GI tract, NO-GC is expressed in several cell types such as smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). Using cell-specific knockout mice, we have previously shown that NO-GC modulates spontaneous contractions in colonic longitudinal smooth muscle. However, its detailed role in the colonic circular smooth muscle is still unclear. Methods Myography was performed to evaluate spontaneous contractions in rings of proximal colon (2.5 mm) from global (GCKO) and cell-specific knockout mice for NO-GC. Immunohistochemistry and in situ hybridization were used to specify NO-GC expression. Key results Colonic circular smooth muscle showed three different contraction patterns: high-frequency ripples, slow phasic contractions, and large contractions. Ripples formed independently of NO-GC. Slow phasic contractions occurred intermittently in WT, SMC-GCKO, and ICC-GCKO tissue, whereas they were more prominent and prolonged in GCKO and SMC/ICC-GCKO tissue. Tetrodotoxin and the NO-GC inhibitor ODQ transformed slow phasic contractions of WT and single cell-specific knockout into GCKO-like contractions. ODQ increased the frequency of large contractions in WT and ICC-GCKO colon but not in GCKO, SMC-GCKO, and SMC/ICC-GCKO preparations. Tetrodotoxin and hexamethonium abolished large contractions. Conclusions and inferences We conclude that short rings of murine colon can be effectively used to record spontaneous contractions. Although NO-GC in SMC determines smooth muscle tone, concerted action of NO-GC in both SMC and ICC modulates slow phasic contractions and large contractions.

Background— Arterial hypertension is one of the major diseases in industrial countries and a major cause of mortality. One of the main vascular factors responsible for the relaxation of blood vessels and regulation of blood pressure is nitric oxide (NO). NO acts predominantly via NO-sensitive guanylyl cyclase (NO-GC), which is made up of 2 different subunits (α and β). Deletion of the β 1 subunit leads to a global NO-GC knockout, and these mice are hypertensive. However, global deletion of NO-GC in mice does not allow identification of the cell/tissue type responsible for the elevated blood pressure. Methods and Results— To determine the relative contribution of smooth muscle cells to the hypertension seen in NO-GC knockout mice, we generated smooth muscle–specific knockout mice for the β 1 subunit of NO-GC using a tamoxifen-inducible system. Male mice were investigated because the Cre transgene used is located on the Y chromosome. Tamoxifen injection led to a rapid reduction of NO-GC expression in smooth muscle but did not affect that in other tissues. Parallel to a reduction in NO-induced cGMP accumulation, NO-induced relaxation of aortic smooth muscle was gradually lost after induction by tamoxifen. Concomitantly, these animals developed hypertension within 3 to 4 weeks. Conclusions— We generated a model in which the development of hypertension can be visualized over time by deletion of a single gene in smooth muscle cells. In sum, our data provide evidence that deletion of NO-GC solely in smooth muscle is sufficient to cause hypertension.

The signaling molecule nitric oxide (NO) acts as physiological activator of NO-sensitive guanylyl cyclase (NO-GC) in the cardiovascular, gastrointestinal and nervous systems. Two isoforms of NO-GC are known to exist on the protein level. The enzyme is a heterodimer consisting of an alpha (alpha(1) or alpha(2)) and a beta subunit (beta(1)). Strategies for the genomic deletion of either subunit have been developed in the recent years. Removal of one of the two isoforms by deletion of one of the alpha subunits allowed the investigation of the specific functions of the respective isoform. The deletion of the beta(1) subunit led to complete knock-out thus completely disrupting the NO/cGMP signaling cascade. The phenotypes of these KO mice have corroborated the already known physiological importance of the NO/cGMP cascade e.g. in the regulation of blood pressure, platelet inhibition, interneuronal communication; yet, they have also given hints to novel functions and mechanisms. In addition, mice lacking both NO-GC isoforms permitted the investigation of possible cGMP-independent signaling pathways of NO. As cell- and tissue-specific knock-out models are beginning to emerge, a more detailed analysis of the importance of the NO receptor in specific tissues will become possible.

The signaling molecule nitric oxide (NO), first described as endothelium-derived relaxing factor (EDRF), acts as physiological activator of NO-sensitive guanylyl cyclase (NO-GC) in the cardiovascular, gastrointestinal, and nervous systems. Besides NO-GC, other NO targets have been proposed; however, their particular contribution still remains unclear. Here, we generated mice deficient for the β 1 subunit of NO-GC, which resulted in complete loss of the enzyme. GC-KO mice have a life span of 3–4 weeks but then die because of intestinal dysmotility; however, they can be rescued by feeding them a fiber-free diet. Apparently, NO-GC is absolutely vital for the maintenance of normal peristalsis of the gut. GC-KO mice show a pronounced increase in blood pressure, underlining the importance of NO in the regulation of smooth muscle tone in vivo . The lack of an NO effect on aortic relaxation and platelet aggregation confirms NO-GC as the only NO target regulating these two functions, excluding cGMP-independent mechanisms. Our knockout model completely disrupts the NO/cGMP signaling cascade and provides evidence for the unique role of NO-GC as NO receptor.

Background In the inner ear, elevated cyclic guanosine 3’-5’-monophospate (cGMP) levels were shown to have a sheltering role for cochlear hair cells and hearing function [1]. However, the individual roles of the two nitric oxide-sensitive guanylyl cyclase isoforms (NO-GC1 and NO-GC2) as cGMP generators in this protective effect are still unclear. The aim of this study was to investigate how the deletion of either one of the a-subunits of NO-GC (NO-GC1 KO or NO-GC2 KO) [2] or deletion of the b1-subunit of NO-GC (NO-GC KO), leading to global lack of NO-GC expression [3], affects hearing function, vulnerability to noise exposure and recovery from acoustic trauma in mice.

Background Chronic pain in response to tissue inflammation (inflammatory pain) or nerve injury (neuropathic pain) is often unresponsive to currently available treatments. A large body of evidence indicates that production of nitric oxide (NO) and activation of NO-sensitive guanylyl cyclase (NO-GC) essentially contributes to the processing of chronic pain. NO-GC is a heterodimer consisting of one a subunit (a1 or a2) and one b1 subunit and exists in two isoforms (NO-GC1 and NOGC2). However, the functional role of NO-GC1 and NO-GC2 in pain processing remains poorly understood. Here, we investigated the expression of NO-GC isoforms in pain-relevant tissues (dorsal root ganglia and the spinal cord) and characterized the nociceptive behavior of mice lacking a1 or a2 in models of acute nociceptive, inflammatory and neuropathic pain.

ObjectivesWe have previously described the antifibrotic role of the soluble guanylate cyclase (sGC). The mode of action, however, remained elusive. In the present study, we describe a novel link between sGC signalling and transforming growth factor β (TGFβ) signalling that mediates the antifibrotic effects of the sGC.MethodsHuman fibroblasts and murine sGC knockout fibroblasts were treated with the sGC stimulator BAY 41-2272 or the stable cyclic guanosine monophosphate (cGMP) analogue 8-Bromo-cGMP and stimulated with TGFβ. sGC knockout fibroblasts were isolated from sGCIfl/fl mice, and recombination was induced by Cre-adenovirus. In vivo, we studied the antifibrotic effects of BAY 41-2272 in mice overexpressing a constitutively active TGF-β1 receptor.ResultssGC stimulation inhibited TGFβ-dependent fibroblast activation and collagen release. sGC knockout fibroblasts confirmed that the sGC is essential for the antifibrotic effects of BAY 41-2272. Furthermore, 8-Bromo-cGMP reduced TGFβ-dependent collagen release. While nuclear p-SMAD2 and 3 levels, SMAD reporter activity and transcription of classical TGFβ target genes remained unchanged, sGC stimulation blocked the phosphorylation of ERK. In vivo, sGC stimulation inhibited TGFβ-driven dermal fibrosis but did not change p-SMAD2 and 3 levels and TGFβ target gene expression, confirming that non-canonical TGFβ pathways mediate the antifibrotic sGC activity.ConclusionsWe elucidated the antifibrotic mode of action of the sGC that increases cGMP levels, blocks non-canonical TGFβ signalling and inhibits experimental fibrosis. Since sGC stimulators have shown excellent efficacy and tolerability in phase 3 clinical trials for pulmonary arterial hypertension, they may be further developed for the simultaneous treatment of fibrosis and vascular disease in systemic sclerosis.

In the vascular system, the receptor for the signaling molecule NO, guanylyl cyclase (GC), mediates smooth muscle relaxation and inhibition of platelet aggregation by increasing intracellular cyclic GMP (cGMP) concentration. The heterodimeric GC exists in 2 isoforms (alpha1-GC, alpha2-GC) with indistinguishable regulatory properties. Here, we used mice deficient in either alpha1- or alpha2-GC to dissect their biological functions. In platelets, alpha1-GC, the only isoform present, was responsible for NO-induced inhibition of aggregation. In aortic tissue, alpha1-GC, as the major isoform (94%), mediated vasodilation. Unexpectedly, alpha2-GC, representing only 6% of the total GC content in WT, also completely relaxed alpha1-deficient vessels albeit higher NO concentrations were needed. The functional impact of the low cGMP levels produced by alpha2-GC in vivo was underlined by pronounced blood pressure increases upon NO synthase inhibition. As a fractional amount of GC was sufficient to mediate vasorelaxation at higher NO concentrations, we conclude that the majority of NO-sensitive GC is not required for cGMP-forming activity but as NO receptor reserve to increase sensitivity toward the labile messenger NO in vivo.

The NO/cGMP signaling pathway plays a major role in the cardiovascular system, in which it is involved in the regulation of smooth muscle tone and inhibition of platelet aggregation. Under pathophysiological conditions such as endothelial dysfunction, coronary artery disease, and airway hyperreactivity, smooth muscle containing arteries and bronchi are of great pharmacological interest. In these tissues, NO mediates its effects by stimulating guanylyl cyclase (GC) to form cGMP; the subsequent increase in cGMP is counteracted by the cGMP-specific phosphodiesterase (PDE5), which hydrolyzes cGMP. In platelets, allosteric activation of PDE5 by cGMP paralleled by phosphorylation has been shown to govern the sensitivity of NO/cGMP signaling. Here, we demonstrate that the functional responsiveness to NO correlates with the relative abundance of GC and PDE5 in aortic and bronchial tissue, respectively. We show a sustained desensitization of the NO-induced relaxation of aortic and bronchial rings caused by a short-term exposure to NO. The NO treatment caused heterologous desensitization of atrial natriuretic peptide-induced relaxation, whereas relaxation by the cGMP analog 8-pCPT-cGMP was unperturbed. Impaired relaxation was shown to be paralleled by PDE5 phosphorylation; this indicates enhanced cGMP degradation as a mechanism of desensitization. In summary, our results demonstrate the physiological impact of PDE5 activation on the control of smooth muscle tone and provide an explanation for the apparent impairment of NO-induced vasorelaxation.

The enzyme nitric oxide-sensitive guanylyl cyclase is an obligate heterodimer consisting of an alpha and a beta subunit. Whereas the C-terminal parts of the subunits have been shown to be sufficient for catalysis, regulation was assigned to the N termini. The central domains have been postulated to be responsible for the formation of alphabeta heterodimers. Here, we have analyzed dimerization by precipitation of various N- and C-terminally truncated alpha(1) mutants with beta(1) wild type or deletion mutants thereof after coexpression in the baculovirus/Sf9 system. In contrast to the current hypothesis, our analysis revealed that an N-terminal region of the alpha(1) subunit (amino acids 61-128) is mandatory for quantitative dimerization. The central domain (amino acids 367-462) contributes but is not sufficient to mediate robust alphabeta interaction. Wild type-like binding of the identified minimum dimerization region of alpha(1) (amino acids 61-462) requires the N-terminal and central region of beta(1) (amino acids 1-385). Furthermore, we observed an unequal stability of the alpha(1) and beta(1) subunit. Whereas beta(1) forms heme containing homodimers and is stable, alpha(1) appears to be prone to misfolding and degradation when heterodimerization is impaired by deletion of important sequences.

By the formation of the second messenger cGMP, NO-sensitive guanylyl cyclase (GC) plays a key role within the NO/cGMP signaling cascade which participates in vascular regulation and neurotransmission. The enzyme contains a prosthetic heme group that acts as the acceptor site for NO. High affinity binding of NO to the heme moiety leads to an up to 200-fold activation of the enzyme. Unexpectedly, NO dissociates with a half-life of a few seconds which appears fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogs act as NO sensitizers and led to the discovery of a novel pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme. The two isoforms of the heterodimeric enzyme (alpha1beta1, alpha2beta1) are known that are functionally indistinguishable. The alpha2beta1-isoform mainly occurs in brain whereas the alpha1beta1-enzyme shows a broader distribution and represents the predominantly expressed form of NO-sensitive GC. Until recently, the enzyme has been thought to occur in the cytosol. However, latest evidence suggests that the alpha2-subunit mediates the membrane association of the alpha2beta1-isoform via interaction with a PDZ domain of the post-synaptic scaffold protein PSD-95. Binding to PSD-95 locates this isoform in close proximity to the NO-generating synthases thereby enabling the NO sensor to respond to locally elevated NO concentrations. In sum, the two known isoforms may stand for the neuronal and vascular form of NO-sensitive GC reflecting a possible association to the neuronal and endothelial NO-synthase, respectively.

1 Carbon monoxide (CO), one of the end products of heme catabolism by heme oxygenase, possesses antihypertensive and vasodilatory characteristics. We have recently discovered that certain transition metal carbonyls are capable of releasing CO in biological fluids and modulate physiological functions via the delivery of CO. Because the initial compounds identified were not water soluble, we have synthesized new CO-releasing molecules that are chemically modified to allow solubility in water. The aim of this study was to assess the vasoactive properties of tricarbonylchloro(glycinato)ruthenium(II) (CORM-3) in vitro and in vivo. 2 CORM-3 produced a concentration-dependent relaxation in vessels precontracted with phenylephrine, exerting significant vasodilatation starting at concentrations of 25-50 microm. Inactive CORM-3, which does not release CO, did not affect vascular tone. 3 Blockers of ATP-dependent potassium channels (glibenclamide) or guanylate cyclase activity (ODQ) considerably reduced CORM-3-dependent relaxation, confirming that potassium channels activation and cGMP partly mediate the vasoactive properties of CO. In fact, increased levels of cGMP were detected in aortas following CORM-3 stimulation. 4 The in vitro and in vivo vasorelaxant activities of CORM-3 were further enhanced in the presence of YC-1, a benzylindazole derivative which is known to sensitize guanylate cyclase to activation by CO. Interestingly, inhibiting nitric oxide production or removing the endothelium significantly decreased vasodilatation by CORM-3, suggesting that factors produced by the endothelium influence CORM-3 vascular activities. 5 These results, together with our previous findings on the cardioprotective functions of CORM-3, indicate that this molecule is an excellent prototype of water-soluble CO carriers for studying the pharmacological and biological features of CO.

Most of the effects of the signalling molecule nitric oxide (NO) are mediated by the activation of NO-sensitive guanylyl cyclase (GC). Subsequent to the NO-induced stimulation of cGMP synthesis, the rise in the intracellular cGMP concentration induces the activation of the different cGMP effector molecules. Accumulating evidence has been presented that the sensitivity of the cGMP response is modulated by the amount of NO present, i.e., a lack of NO was shown to lead to an enhanced cGMP response in aortas in response to NO stimulation while preincubation with NO blunted this cGMP response. Here, we show that l - N -nitro-arginine-methyl ester ( l -NAME) treatment of rats leads to a very much increased cGMP response toward sodium nitroprusside (SNP) in aortic tissue. In the aortic cytosolic fraction, enzyme activities of GC and phosphodiesterase (PDE) did not differ between the two animal groups. We did not detect any difference in the expression of NO-sensitive GC between l -NAME-treated and control animals, which could explain the enhanced NO response. The results show that a reduction of the endogenous NO production induced by long-term l -NAME treatment does not lead to an up-regulation of NO-sensitive GC on the level of protein expression.

In platelets, the nitric oxide (NO)–induced cGMP response is indicative of a highly regulated interplay of cGMP formation and cGMP degradation. Recently, we showed that within the NO-induced cGMP response in human platelets, activation and phosphorylation of phosphodiesterase type 5 (PDE5) occurred. Here, we identify cyclic GMP-dependent protein kinase I as the kinase responsible for the NO-induced PDE5 phosphorylation. However, we demonstrate that cGMP can directly activate PDE5 without phosphorylation in platelet cytosol, most likely via binding to the regulatory GAF domains. The reversal of activation was slow, and was not completed after 60 min. Phosphorylation enhanced the cGMP-induced activation, allowing it to occur at lower cGMP concentrations. Also, in intact platelets, a sustained NO-induced activation of PDE5 for as long as 60 min was detected. Finally, the long-term desensitization of the cGMP response induced by a low NO concentration reveals the physiological relevance of the PDE5 activation within NO/cGMP signaling. In sum, we suggest NO-induced activation and phosphorylation of PDE5 as the mechanism for a long-lasting negative feedback loop shaping the cGMP response in human platelets in order to adapt to the amount of NO available.

Nitric oxide (NO) acts as essential regulator of vasculogenesis and angiogenesis and is critical for arteriogenesis. Whether NO's effects in vivo are mediated through NO-sensitive guanylyl cyclase (NO-GC) and thus by cGMP-dependent mechanisms has been only poorly addressed. Mice lacking NO-GC globally or specifically in smooth muscle cells (SMC) or endothelial cells (EC) were subjected to two established models for arteriogenesis and angiogenesis, namely hindlimb ischemia and oxygen-induced retinopathy. Our data clearly show the involvement of NO-GC in the recovery of blood flow after hindlimb ischemia, and this effect could be attributed to NO-GC in SMC. In the retina, global deletion of NO-GC led to reduced oxygen-induced vessel loss and hypoxia-induced capillary regrowth, whereas pathological neovascularization was increased. These effects were also seen in mice with SMC-specific NO-GC deletion but not in animals lacking NO-GC in EC. Intriguingly, NO-GC was found to be strongly expressed in retinal pericytes. Our data prove the involvement of NO-GC in growth and plasticity of hindlimb and retinal vasculature after ischemic/hypoxic insult.

Background Nitric oxide (NO) is an important inhibitory neurotransmitter in the gastrointestinal (GI) tract. The majority of nitrergic effects are transduced by NO-sensitive guanylyl cyclase (NO-GC) as the receptor for NO, and, thus, mediated by cGMP-dependent mechanisms. Work carried out during the past years has demonstrated NO to be largely involved in GI smooth muscle relaxation and motility. However, detailed investigation of nitrergic signaling has turned out to be complicated as NO-GC was identified in several different GI cell types such as smooth muscle cells, interstitial cells of Cajal and fibroblast-like cells. With regards to nitrergic neurotransmission, special focus has been placed on the role of interstitial cells of Cajal using mutant mice with reduced populations of ICC. Recently, global and cell-specific knockout mice for enzymes participating in nitrergic signaling have been generated providing a suitable approach to further examine the role of NO-mediated signaling in GI smooth muscle. Purpose This review discusses the current knowledge on nitrergic mechanisms in gastrointestinal neuromuscular transmission with a focus on genetic models and outlines possible further investigations to gain better understanding on NO-mediated effects in the GI tract.

Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.

In previous studies, a strong synergism between low concentrations of hydrogen peroxide and nitric oxide in the inhibition of agonist-induced platelet aggregation has been established and may be due to enhanced formation of cyclic GMP. In this investigation, hydrogen peroxide and NO had no effect on the activity of pure soluble guanylyl cyclase or its activity in platelet lysates and cytosol. H(2)O(2) was found to increase the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), increasing the amount of the 50-kDa form that results from phosphorylation at serine(157). This occurs both in the presence and in the absence of low concentrations of NO, even at submicromolar concentrations of the peroxide, which alone was not inhibitory to platelets. These actions of H(2)O(2) were inhibited to a large extent by an inhibitor of cyclic AMP-dependent protein kinase, even though H(2)O(2) did not increase cyclic AMP. This inhibitor reversed the inhibition of platelets induced by combinations of NO and H(2)O(2) at low concentrations. The results suggest that the action on VASP may be one site of action of H(2)O(2) but that this event alone does not lead to inhibition of platelets; another unspecified action of NO is required to complete the events required for inhibition.

We used YC-1 as a pharmacological tool to investigate the short-term blood pressure effects of NO-independent activation of sGC in normotensive and hypertensive rats. Four groups of normotensive Wistar-Kyoto rats were treated by i.v. injection with vehicle (V), YC-1 (YC-1), sodium nitroprusside (SNP), or YC-1 and SNP (YC-1+SNP). Hypertension was induced in four additional groups of WKY rats by 3 weeks of oral treatment with L-NAME. These animals were investigated with the same protocol as the normotensive animals: L-NAME/V, L-NAME/YC-1, L-NAME/SNP, L-NAME/YC-1+SNP. YC-1 lowered mean arterial blood pressure (MAP) in normotensive and hypertensive animals similarly to SNP alone (P

Analysis of purified soybean and rabbit reticulocyte 15-lipoxygenase (15-LOX) and PA317 cells transfected with human 15-LOX revealed a rapid rate of linoleate-dependent nitric oxide (.NO) uptake that coincided with reversible inhibition of product ((13S)-hydroperoxyoctadecadienoic acid, or (13S)-HPODE) formation. No reaction of .NO (up to 2 microM) with either native (Ered) or ferric LOXs (0.2 microM) metal centers to form nitrosyl complexes occurred at these .NO concentrations. During HPODE-dependent activation of 15-LOX, there was consumption of 2 mol of .NO/mol of 15-LOX. Stopped flow fluorescence spectroscopy showed that.NO (2.2 microM) did not alter the rate or extent of (13S)-HPODE-induced tryptophan fluorescence quenching associated with 15-LOX activation. Additionally, .NO does not inhibit the anaerobic peroxidase activity of 15-LOX, inferring that the inhibitory actions of .NO are due to reaction with the enzyme-bound lipid peroxyl radical, rather than impairment of (13S)-HPODE-dependent enzyme activation. From this, a mechanism of 15-LOX inhibition by .NO is proposed whereby reaction of .NO with EredLOO. generates Ered and LOONO, which hydrolyzes to (13S)-HPODE and nitrite (NO2-). Reactivation of Ered, considerably slower than dioxygenase activity, is then required to complete the catalytic cycle and leads to a net inhibition of rates of (13S)-HPODE formation. This reaction of .NO with 15-LOX inhibited. NO-dependent activation of soluble guanylate cyclase and consequent cGMP production. Since accelerated .NO production, enhanced 15-LOX gene expression, and 15-LOX product formation occurs in diverse inflammatory conditions, these observations indicate that reactions of .NO with lipoxygenase peroxyl radical intermediates will result in modulation of both .NO bioavailability and rates of production of lipid signaling mediators.

Soluble guanylate cyclase (sGC), which is found in many cells and tissues, represents the receptor for the intra- and intercellular messenger molecule NO. Superoxide dismutase (SOD), an enzyme involved in the degradation of toxic superoxide radicals, has been proposed as a non-NO activator of sGC. Here we show that SOD stimulated sGC purified from bovine lung up to 10-fold. Activation by SOD was not influenced by the hydroxyl radical scavengers mannitol and DMSO. In contrast, the presence of the NO scavengers oxyhaemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, as well as the O2--generating system xanthine oxidase/hypoxanthine, led to inhibition of SOD-stimulated cGMP production. NO-insensitive sGC mutants were not influenced either by SOD or by xanthine oxidase. We have previously shown that sGC was stimulated by NO present in the normal atmosphere. Here we show that the SOD effect depended on the NO concentration from the atmosphere, as the stimulation of sGC by defined NO gases (0, 120, 330 and 1000 parts per billion NO) was potentiated by SOD. NO stimulation of sGC and its potentiation by SOD were inhibited by oxyhaemoglobin to identical levels. We conclude that the SOD-mediated stimulation of sGC is due to the elimination of superoxide, thereby preventing its reaction with NO to form peroxynitrite.

Soluble guanylyl cyclase (sGC), a heme-containing heterodimeric enzyme, is stimulated by NO and catalyzes the formation of the intracellular signaling molecule cGMP. Cysteine residues of sGC have been considered to be important as they were thought to play a significant role in the regulation of the enzyme. The aim of this study was to investigate the possible function of conserved cysteine residues of sGC. Fifteen conserved cysteine residues on sGC were point-mutated to serine, using site-directed mutagenesis. All of the resulting recombinant enzymes were able to synthesize cGMP. Mutation of two cysteines located in the N-terminal, putative heme-binding region of the beta1 subunit yielded proteins that were insensitive to NO. Spectrophotometric analysis of the NO-insensitive mutants purified from Sf9 cells revealed a loss of the prosthetic heme group. Both mutants could be reconstituted with heme and, as a consequence, NO sensitivity of the mutants was restored. Our data show that mutation of two cysteines of the beta1 subunit (Cys-78 and Cys-214) reduces the affinity of sGC for heme. Mutation of the corresponding cysteines on the alpha1 subunit did not alter NO responsiveness, indicating that heme-binding is mainly a feature of the N-terminal domain of the beta1 subunit.

Background There are two types of adipose tissue. White adipose tissue (WAT) stores energy in the form of lipids. Brown adipose tissue (BAT) consumes energy to produce heat by thermogenesis, which depends on the brown adipocytes specific marker uncoupling protein-1 (UCP—1). Metabolically active BAT is present in adult humans and its energy consuming properties could be exploited to increase energy expenditure. We and others showed that the nitric oxide (NO)/ cyclic guanosine monophosphate (cGMP) pathway is crucial for brown adipocyte differentiation [1,2]. Here we studied the role of sGC in brown adipocyte differentiation employing the sGC stimulator BAY 41—2272. Material and methods Mesenchymal stem cells isolated from BAT of newborn mice were differentiated into brown adipocytes in vitro using established protocols [1]. During differentiation, cells were incubated with 3 µM BAY 41—2272, 200 µM 8pCPT-cGMP or 30 µM ODQ. Accumulated intracellular lipids were assessed by RedO staining and measurement of triglyceride (TG) content. To investigate the adipogenic program, Western Blot analysis of PPARg ,C /EBPa and AP2 was performed. Thermogenic differentiation of the cells was determined by protein expression analysis of Cytc and gene expression analysis of thermogenic markers UCP—1, Cidea and PGC-1a. Results BAY 41-2272 increased lipid accumulation as determined by RedO staining and TG content (1,44-fold) compared to control. Protein expression of the adipogenic marker proteins PPARg (1,4-fold), C/EBPa (1,5-fold) and AP2 (1,6—fold) were increased after BAY 41-2272 incubation compared to control. Adipogenic markers PPARg (2,3-fold), C/EBPa (1,8-fold) and AP2 (2,1-fold) were increased after cGMP incubation compared to control. In contrast, inhibition of sGC by ODQ decreased protein expression of PPARg (50%), C/EBPa (29%) and AP2 (51%). The thermogenic marker genes UCP-1 (5,38-fold), Cidea (2,44-fold) and PGC-1a (2,26fold) increased upon incubation with BAY 41-2272 compared to control. cGMP increased RNA levels of thermogenic markers UCP—1 (3,36-fold), Cidea (2,5fold) and PGC-1a (2,50 fold). ODQ decreased the expression of UCP-1 (64%), Cidea (74%) and PGC—1a (44%). Protein expression of the mitochondrial marker Cytc was increased upon incubation with BAY 41—2272 (1,6-fold) and cGMP (1,77-fold). Protein expression of Cytc was lower in brown adipocytes incubated with ODQ (49%). Conclusion

Background Nitric oxide (NO) is well known to stimulate NO-sensitive guanylyl cyclase (NO-GC) followed by production of cGMP and, eventually, to cause smooth muscle relaxation. Recent studies have shown that NO-mediated relaxation of gastrointestinal tissue is dependent on NO-GC in at least two cell types: smooth muscle cells (SMC) and interstitial cells of Cajal (ICC). In addition, the role of NO-GC in a novel interstitial cell type called fibroblast-like cells (FLC) in enteric neurotransmission is debated. In contrast, information about distribution and function of NO-GC in the lower urinary (LU) tract which comprises bladder and urethra is sparse. Here, we examined the function of NOGC in smooth muscle of the gastrointestinal (GI) and the LU tract.

Chronic challenge of renin–angiotensin causes recruitment of renin-producing cells in the kidney along the media layer of afferent arterioles and hypertrophy of cells in the juxtaglomerular apparatus. This study aimed to define the role of nitric oxide (NO) with regard to the recruitment pattern of renin-producing cells and to the possible pathways along which NO could act. We considered the hypothesis that endothelium-derived NO acts via NO-sensitive guanylate cyclase. Mice were treated with low-salt diet in combination with the angiotensin I–converting enzyme inhibitor enalapril for 3 weeks, which led to a 13-fold increase in renin expression associated with marked recruitment of renin cells in afferent arterioles and hypertrophy of the juxtaglomerular apparatus in wild-type mice. In wild-type mice additionally treated with the nonselective NO synthase inhibitor L-NAME, the recruitment of renin-expressing cells along the afferent arterioles was absent and juxtaglomerular hypertrophy was diminished. An almost identical attenuation of renin cell recruitment as with L-NAME treatment in wild-type mice was found in mice lacking the endothelial isoform of NO synthase. Treatment of mice lacking NO-sensitive guanylate cyclase in renin-expressing cells and preglomerular smooth muscle cells with low-salt diet in combination with the angiotensin I–converting enzyme inhibitor enalapril for 3 weeks produced juxtaglomerular hypertrophy like in wild-type mice, but no recruitment in afferent arterioles. These findings suggest that endothelium-derived NO and concomitant formation of cGMP in preglomerular renin cell precursors supports recruitment of renin-expressing cells along preglomerular vessels, but not in the juxtaglomerular apparatus.